Ddx3 as a biomarker for cancer and methods related thereto

ABSTRACT

The invention encompasses methods for treating or preventing diseases and disorders associated abnormal cell growth, for example, treating or preventing cancer or tumor growth, by administering to a subject in need thereof a composition comprising a therapeutically or prophylactically effective amount of a compound that downregulates DDX3, for example a fused diimidazodiazepine ring compound or a pharmaceutically acceptable salt thereof. The invention also encompasses the use of DDX3 as a biomarker for diagnostic and treatment purposes, for example, to identify a hyperproliferative disorder susceptible to treatment by down regulation of DDX3.

This application is a divisional of U.S. application Ser. No.13/840,549, which is a continuation-in-part of U.S. application Ser. No.13/061,612, which is a 371 National Stage of PCT InternationalApplication No. PCT/US2009/05273, filed Sep. 23, 2009, which claims thebenefit of U.S. provisional patent application No. 61/099,324, filedSep. 23, 2008, the disclosures of which are incorporated herein byreference in their entirety. This application is also acontinuation-in-part of PCT International Application No.PCT/US2012/028475, filed Mar. 9, 2012, which claims the benefit of U.S.provisional patent application No. 61/450,969, filed Mar. 9, 2011, thedisclosures of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The invention encompasses methods for identifying hyperproliferativedisorders and subjects having hyperproliferative disorders treatable bydownregulation of DDX3 by determining expression of DDX3 in tissues andcells. The invention also encompasses methods of treatinghyperproliferative disorders by administering a compound thatdownregulates DDX3 to a subject having a hyperproliferative disordersthat overexpresses DDX3. The invention also encompasses methods fortreating or preventing cancer, tumors and other disorders associatedabnormal cell growth by administering to a subject in need thereof acomposition comprising a therapeutically or prophylactically effectiveamount of a compound that downregulates DDX3. The invention alsoencompasses methods for treating hyperproliferative disorders usingcompounds having a fused diimidazodiazepine ring system thatdownregulates DDX3.

BACKGROUND OF THE INVENTION

Treatment of cancer varies based on the type of cancer and its stage.The stage of a cancer refers to how much it has grown and whether thetumor has spread from its original location. If the cancer is confinedto one location and has not spread, the most common goals for treatmentare surgery and cure. This is often the case with skin cancers, as wellas cancers of the lung, breast, and colon.

If the tumor has spread to local lymph nodes only, sometimes these canalso be removed. If surgery cannot remove all of the cancer, the optionsfor treatment include radiation, chemotherapy, or both. Some cancersrequire a combination of surgery, radiation, and chemotherapy.

Although current radiotherapeutic agents, chemotherapeutic agents andbiological toxins are potent cytotoxins, they do not discriminatebetween normal and malignant cells, producing adverse effects anddose-limiting toxicities. There remains specific cancer markers andidentifying cancers, tumors and other disorders associated abnormal cellgrowth that can be treated with drugs that are effective for treatingsuch disorders without producing adverse effects and dose-limitingtoxicities to normal cells.

Surprisingly, the methods and compositions of the invention fulfill theneeds and satisfy other objects and advantages that will become apparentfrom the description which follows.

SUMMARY OF THE INVENTION

The present invention relates to methods of treating cancer and otherhyperproliferative disorders using compounds that are effective indiscriminately inhibiting the growth of cancer and tumor cells bydownregulation of DDX3 while allowing healthy “normal” cells to remainunaffected. The present invention also relates to methods foridentifying cancers, tumors and other hyperproliferative disordersassociated with abnormal cell growth treatable by the specificallydescribed compounds and others.

The invention encompasses methods of treatment by administeringcompounds that downregulate expression of DDX3, including the compoundsof Formulas I-V, that are useful for treating or preventing a disease ordisorder including, but not limited to, conditions caused byuncontrolled cell growth, hyperproliferation of cells, tumor growth, andcancers.

The invention also encompasses methods for treating or preventingpancreatic cancer, liver cancer, ovarian cancer, breast cancer; lungcancer; prostate cancer; brain cancer including glioblastoma,medulloblastoma, and pontine tumors; kidney cancer; leukemia; or asarcoma, which comprises administering to a mammal in need of suchtreatment or prevention a therapeutically or prophylactically effectiveamount of a composition comprising a compound of Formula I-V, or apharmaceutically acceptable salt or prodrug thereof, and apharmaceutically acceptable vehicle.

DDX3, a member of a RNA helicase family, is dysregulated in many cancertypes including, breast cancer; lung cancer; prostate cancer; braincancer including glioblastoma, medulloblastoma, and pontine tumors;kidney cancer; leukemia; and a sarcomas. The present invention providesthat that targeted downregulation of the expression of DDX3, using asmall molecule inhibitor, for example a fused diimidazodiazepine, iseffective on lung cancer cell lines including a carboplatin resistantcell line.

In accordance with an embodiment, the present invention provides thatcompounds, for example a fused diimidazodiazepine compound according tothe invention, or a salt, solvate, stereoisomer, or derivative thereof,and a pharmaceutically acceptable carrier, acting as a DDX3 inhibitor ina mammalian cell or population of cells in a subject suffering from aproliferative disease are useful in treating the hyperproliferativedisorder.

In accordance with another embodiment, the present invention provides amethod of treating cancer in a subject comprising administering to thesubject an effective amount of a composition comprising a compound thatis a DDX3 inhibitor, for example, a fused diimidazodiazepine compoundaccording to the invention, or a salt, solvate, stereoisomer, orderivative thereof, and a pharmaceutically acceptable carrier.

The invention includes a method of treating a hyperproliferativedisorder susceptible to treatment by downregulation of DDX3 byadministering to a subject in need thereof an effective amount of acomposition or formulation comprising a compound that downregulatesDDX3. Compounds that downregulate DDX3 include compounds of Formula (I):

or pharmaceutically acceptable salts and prodrugs thereof, as describedfurther below. A specific compound is

The hyperproliferative disorder can be, for example breast cancer, lungcancer, prostate cancer, glioblastoma, kidney cancer leukemia,medulloblastoma, pontine tumors, or a sarcoma. Specific disordersinclude breast cancer, lung cancer, prostate cancer or glioblastoma. Thesarcoma can be, for example, chondrosarcoma, malignant fibroushistiocytoma (MFH), clear cell sarcoma, malignant peripheral nervesheath tumor (MPNST), epithelioid sarcoma, myxoid sarcomas, EwingsSarcoma (ESFT or PNET), Kaposi's sarcoma, leiomyosarcoma, pleomorphicsarcoma, fibrosarcoma, low-grade sarcoma and rhabdomyosarcoma (RMS).

The invention is also a method of identifying a subject having ahyperproliferative disorder susceptible to treatment by down regulationof DDX3. The method includes the steps of obtaining a cell or tissuesample from tissue of the subject associated with or suspected to beassociated with the disorder; optionally isolating cells from thetissue; measuring expression of DDX3 in the tissue or in the cellsisolated from the tissue; and identifying the subject as having thehyperproliferative disorder as susceptible to treatment by downregulation of DDX3 if DDX3 is expressed in the tissue or cell sample.The hyperproliferative disorder can be, for example, breast cancer; lungcancer; prostate cancer; brain cancer including glioblastoma,medulloblastoma, and pontine tumors; kidney cancer; leukemia; or asarcoma. In some embodiments, the hyperproliferative disorder is breastcancer, lung cancer, prostate cancer or glioblastoma. In otherembodiments the hyperproliferative disorder is breast cancer. Inembodiments, the hyperproliferative disorder is lung cancer. MeasuringDDX3 expression can be accomplished by immunohistochemistry analysis ofthe tissue sample. Measuring DDX3 expression also can be accomplished byisolating cells from the tissue sample and measuring expression of DDX3in the tissue by measuring expression of DDX3 in the cells. The methodcan include the steps of performing an assay by exposing cells to acompound that downregulates DDX3, measuring cell viability as a functionof concentration and identifying the hyperproliferative disorder assusceptible to treatment by down regulation of DDX3 if cell viabilitydecreases as a function of concentration. The assay can be a MTS assay.The compound that deregulates DDX3 expression can be a compound ofFormula (I) as described herein, for example:

The present invention may be understood more fully by reference to thefigures, detailed description, and examples, which are intended toexemplify non-limiting embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the viability of normal lung cell lines whencontacted with illustrative compound of the invention;

FIGS. 2A and 2B show immunoblots depicting DDX3 expression in lungcancer cell lines. 2A) DDX3 expression in Beas2B cells exposed to twodifferent concentrations of cigarette smoke condensate (CSC). FIG. 2B)DDX3 expression in non-small cell lung cancer cell lines (H23 and A549).Actin was used as a loading control;

FIG. 3 is a bar graph of the number of colonies formed in soft agarassays using A549 and A549-shDDX3 cells. Experiments were done intriplicate;

FIG. 4 is a set of photomicrographs depicting expression of DDX3 innormal lung and in different pathological stages of lung cancer;

FIG. 5 is a chart showing Compound 3 inhibits helicase activity of DDX3;

FIG. 6 is a plot showing the effect of Compound 3 on two lung cancercell lines. Lung cancer cell lines were incubated with differentconcentrations of Compound 3. At day 3, MTS assays were performed todetermine the percentage of metabolically active cells. The experimentswere repeated in triplicate. The IC50 values for H23 and A549 are 3.5and 5 μM respectively;

FIG. 7 illustrates the viability of lung cancer cell lines A549 and H23when contacted with illustrative compound of the invention;

FIG. 8 shows that Compound 3 can induce cell death under hypoxicconditions. Cells engineered to express a hypoxia induced redfluorescent protein (tdTomato) were incubated with CoCl₂ (hypoxiamimetic compound) for 24 hours, following which Compound 3 was added andincubated for 72 hours. Photomicrographs were taken at 20×. Experimentswere repeated twice.

FIG. 9 illustrates DDX3 expression in normal breast cell lines (1-2) anda series of breast cancer cell lines (3-7): FIG. 9A) qRT-PCR of DDX3levels in a series of immortalized normal breast cell lines (1-2) andbreast cancer cell lines (3-7). The breast cancer cell lines are in theorder of aggressive phenotype. FIG. 9B) Immunoblot analysis for DDX3expression in the identical cell lines as above.

FIG. 10 illustrates qRT-PCR of DDX3 levels in different grades of humanbreast carcinoma samples (FIG. 10A) and immunostaining for DDX3 levelsin normal mammary epithelium and primary breast carcinomas (FIG. 10B);

FIG. 11 illustrates representative photomicrographs of sections ofbreast tissue stained with DDX3-specific, affinity purified, rabbitpolyclonal antibody.

FIG. 12A is a graph showing tumor growth rate in the mammary fat pad ofSCID mice (preclinical breast cancer model) using wild type and DDX3knockdown MDA-MB-231 cells. FIG. 12B shows photomicrographs of crosssection of lungs of animals injected orthotopically (mammary fat pad)with MDA-MB-231 and MDAMB-231-shDDX3 cells;

FIG. 13 illustrates MTS assays of MCF 10A cells (immortalized normalbreast cell line) incubated with Compound 3 of the invention. X-axisindicates concentration of drug used. The cells were incubated for threedays (fresh drug was added daily) following which cell viability wasdetermined;

FIG. 14 illustrates MTS assays of MCF 12A cells (immortalized normalbreast cell line) incubated with Compound 3 of the invention. X-axisindicates concentration of drug used. The cells were incubated for threedays (fresh drug was added daily) following which cell viability wasdetermined;

FIG. 15 illustrates MTS assays of MCF-7 cells (breast cancer cell line)incubated with Compound 3 of the invention. X-axis indicatesconcentration of drug used. The cells were incubated for three days(fresh drug was added daily) following which cell viability wasdetermined;

FIG. 16 illustrates MTS assays of MDA-MB-468 cells (breast cancer cellline) incubated with Compound 3 of the invention. X-axis indicatesconcentration of drug used. The cells were incubated for three days(fresh drug was added daily) following, which cell viability wasdetermined;

FIG. 17 illustrates MTS assays of MDA-MB-231 cells (breast cancer cellline) incubated with Compound 3 of the invention. X-axis indicatesconcentration of drug used. The cells were incubated for three days(fresh drug was added daily) following which cell viability wasdetermined;

FIG. 18 illustrates MTS assays of HL60T cells (leukemia cell line)incubated with Compound 3 of the invention. X-axis indicatesconcentration of drug used. The cells were incubated for three days(fresh drug was added daily) following which cell viability wasdetermined.

FIG. 19 illustrates MTS assays of HNT34 cells (leukemia cell line)incubated with Compound 3 of the invention. X-axis indicatesconcentration of drug used. The cells were incubated for three days(fresh drug was added daily) following which cell viability wasdetermined.

FIG. 20 illustrates MTS assays of KG101 cells (leukemia cell line)incubated with Compound 3 of the invention. X-axis indicatesconcentration of drug used. The cells were incubated for three days(fresh drug was added daily) following which cell viability wasdetermined.

FIG. 21 illustrates MTS assays of U87 cells (glioblastoma human cellline) incubated with Compound 3 of the invention;

FIG. 22 illustrates MTS assays of PC3 cells (prostate cancer cell line)incubated with Compound 3 of the invention;

FIG. 23 illustrates photomicrographs of normal tissue sections andprostate carcinoma tissue sections stained with DDX3 antibody;

FIG. 24 illustrates photomicrographs of chondrosarcoma and pleomorphicsarcoma tissue sections stained with DDX3 antibody;

FIG. 25 shows exemplary photomicrographs of normal brain tissue,glioblastoma and medulloblastoma;

FIG. 26 illustrates immunoblot analyses for DDX-3 expression in lung,prostate, and glioblastoma cell lines;

FIGS. 27-31 illustrate Toxicity studies of Compound 3 in SCID mouse.Data shown is for control and untreated samples of brain (cerebellum)(FIG. 27), kidney (FIG. 28), Liver (FIG. 29), spleen (FIG. 30), andblood (FIG. 31); and

FIG. 32 is a set of curves illustrating the biodistribution of Compound3 in mice at different time intervals using an LC/MS/MS method. A totalof five mice per time point were used for this study.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein and unless otherwise indicated, the term “alkoxy group”means an —O— alkyl group, wherein alkyl is as defined herein. An alkoxygroup can be unsubstituted or substituted with one or two suitablesubstituents. Preferably, the alkyl chain of an alkyloxy group is from 1to 6 carbon atoms in length, referred to herein, for example, as“(C₁-C₁₀)alkoxy.”

As used herein and unless otherwise indicated, the term “alkenyl group”means a monovalent unbranched or branched hydrocarbon chain having oneor more double bonds therein. The double bond of an alkenyl group can beunconjugated or conjugated to another unsaturated group. Suitablealkenyl groups include, but are not limited to (C₂-C₈)alkenyl groups,such as vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl,pentadienyl, hexadienyl, 2-ethylhexenyl, 2-propyl-2-butenyl,4-(2-methyl-3-butene)-pentenyl. An alkenyl group can be unsubstituted orsubstituted with one or two suitable substituents.

As used herein and unless otherwise indicated, the term “alkylalkoxy” or“alkyloxyalkyl group” means a saturated, monovalent unbranched orbranched hydrocarbon chain covalently bonded to an oxygen and covalentlybonded to a second a saturated, monovalent unbranched or branchedhydrocarbon chain (e.g., -alkyl-O-alkyl).

As used herein and unless otherwise indicated, the term “alkyl” or“alkyl group” means a substituted or unsubstituted, saturated,monovalent unbranched or branched hydrocarbon chain. Examples of alkylgroups include, but are not limited to, (C₁-C₁₀)alkyl groups, such asmethyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl,2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl,2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl,4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl,2-ethyl-1-butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl,and hexyl, and longer alkyl groups, such as heptyl, and octyl. An alkylgroup can be unsubstituted or substituted with one or two suitablesubstituents.

As used herein and unless otherwise indicated, the term “alkynyl group”means monovalent unbranched or branched hydrocarbon chain having one ormore triple bonds therein. The triple bond of an alkynyl group can beunconjugated or conjugated to another unsaturated group. Suitablealkynyl groups include, but are not limited to, (C₂-C₆)alkynyl groups,such as ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl,4-methyl-1-butynyl, 4-propyl-2-pentynyl, and 4-butyl-2-hexynyl. Analkynyl group can be unsubstituted or substituted with one or twosuitable substituents.

As used herein and unless otherwise indicated, the term “aryl group”means a monocyclic or polycyclic-aromatic radical comprising carbon andhydrogen atoms. Examples of suitable aryl groups include, but are notlimited to, phenyl, tolyl, anthacenyl, fluorenyl, indenyl, azulenyl, andnaphthyl, as well as benzo-fused carbocyclic moieties such as5,6,7,8-tetrahydronaphthyl. An aryl group can be unsubstituted orsubstituted with one or two suitable substituents. Preferably, the arylgroup is a monocyclic ring, wherein the ring comprises 6 carbon atoms,referred to herein as “(C₆)aryl.”

As used herein and unless otherwise indicated, the term “aryloxy group”means an —O-aryl group, wherein aryl is as defined herein. An aryloxygroup can be unsubstituted or substituted with one or two suitablesubstituents. Preferably, the aryl ring of an aryloxy group is amonocyclic ring, wherein the ring comprises 6 carbon atoms, referred toherein as “(C₆)aryloxy.”

As used herein, the term “benzyl” means —CH₂-phenyl.

As used herein, the term “carbonyl” group is a divalent group of theformula —C(O)—.

As used herein and unless otherwise indicated, the term “compounds ofthe invention” means, collectively, the compounds of formulas I, II,III, IV, and V and pharmaceutically acceptable salts thereof as well ascompounds depicted herein including Compounds 3, 11, 12, 13, 14, 21, 23,25, 27, and 101-193. The compounds of the invention are identifiedherein by their chemical structure and/or chemical name. Where acompound is referred to by both a chemical structure and a chemicalname, and that chemical structure and chemical name conflict, thechemical structure is determinative of the compound's identity. Thecompounds of the invention may contain one or more chiral centers and/ordouble bonds and, therefore, exist as stereoisomers, such as double-bondisomers (i.e., geometric isomers), enantiomers, or diastereomers.According to the invention, the chemical structures depicted herein, andtherefore the compounds of the invention, encompass all of thecorresponding compound's enantiomers and stereoisomers, that is, boththe stereomerically pure form (e.g., geometrically pure,enantiomerically pure, or diastereomerically pure) and enantiomeric andstereoisomeric mixtures. Enantiomeric and stereoisomeric mixtures can beresolved into their component enantiomers or stereoisomers by well knownmethods, such as chiral-phase gas chromatography, chiral-phase highperformance liquid chromatography, crystallizing the compound as achiral salt complex, or crystallizing the compound in a chiral solvent.Enantiomers and stereoisomers can also be obtained from stereomerically-or enantiomerically-pure intermediates, reagents, and catalysts by wellknown asymmetric synthetic methods.

As used herein and unless otherwise indicated, the term “cylclic alkyl”and “cycloalkyl group” are used synonymously and each means a monocyclicor polycyclic saturated ring comprising carbon and hydrogen atoms andhaving no carbon-carbon multiple bonds. Examples of cycloalkyl groupsinclude, but are not limited to, (C₃-C₇)cycloalkyl groups, such ascyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl, andsaturated cyclic and bicyclic terpenes. A cycloalkyl group can beunsubstituted or substituted by one or two suitable substituents.Preferably, the cycloalkyl group is a monocyclic ring or bicyclic ring.

As used herein and unless otherwise indicated, the term “halogen” meansfluorine, chlorine, bromine, or iodine. Correspondingly, the meaning ofthe terms “halo” and “Hal” encompass fluoro, chloro, bromo, and iodo.

As used herein and unless otherwise indicated, the term “formulation”refers to a composition comprising a compound of the invention that isdescribed in a particular dosage form (e.g., tablet) or with aparticular dosage amount (e.g., 30 mg/kg).

As used herein and unless otherwise indicated, the term “heteroarylgroup” means a monocyclic- or polycyclic aromatic ring comprising carbonatoms, hydrogen atoms, and one or more heteroatoms, preferably 1 to 3heteroatoms, independently selected from nitrogen, oxygen, and sulfur.Illustrative examples of heteroaryl groups include, but are not limitedto, pyridinyl, pyridazinyl, pyrimidyl, pyrazyl, triazinyl, pyrrolyl,pyrazolyl, imidazolyl, (1,2,3,)- and (1,2,4)-triazolyl, pyrazinyl,pyrimidinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, furyl,phenyl, isoxazolyl, and oxazolyl. A heteroaryl group can beunsubstituted or substituted with one or two suitable substituents.Preferably, a heteroaryl group is a monocyclic ring, wherein the ringcomprises 2 to 5 carbon atoms and 1 to 3 heteroatoms, referred to hereinas “(C₂-C₅)heteroaryl.”

As used herein and unless otherwise indicated, the term“heterocycloalkyl group” means a monocyclic or polycyclic ringcomprising carbon and hydrogen atoms and at least one heteroatom,preferably, 1 to 3 heteroatoms selected from nitrogen, oxygen, andsulfur, and having no unsaturation. Examples of heterocycloalkyl groupsinclude pyrrolidinyl, pyrrolidino, piperidinyl, piperidino, piperazinyl,piperazino, morpholinyl, morpholino, thiomorpholinyl, thiomorpholino,and pyranyl. A heterocycloalkyl group can be unsubstituted orsubstituted with one or two suitable substituents. Preferably, theheterocycloalkyl group is a monocyclic or bicyclic ring, morepreferably, a monocyclic ring, wherein the ring comprises from 3 to 7carbon atoms and form 1 to 3 heteroatoms, referred to herein as(C₁-C₇)heterocycloalkyl.

As used herein and unless otherwise indicated, the term “heterocyclicradical” or “heterocyclic ring” means a heterocycloalkyl group or aheteroaryl group.

As used herein and unless otherwise indicated, the term “hydrocarbylgroup” means a monovalent group selected from (C₁-C₈)alkyl,(C₂-C₈)alkenyl, and (C₂-C₈)alkynyl, optionally substituted with one ortwo suitable substituents. Preferably, the hydrocarbon chain of ahydrocarbyl group is from 1 to 6 carbon atoms in length, referred toherein as “(C₁-C₆)hydrocarbyl.”

When administered to a subject (e.g., to an animal for veterinary use orto a human for clinical use), the compounds of the invention can beoptionally administered in isolated form. As used herein, “isolated”means that the compounds of the invention are separated from othercomponents of either (a) a natural source, such as a plant or cell,preferably bacterial culture, or (b) a synthetic organic chemicalreaction mixture, preferably, via conventional techniques, the compoundsof the invention are purified. As used herein, “purified” means thatwhen isolated, the isolate contains at least 80% preferably at least90%, more preferably at least 95%, and most preferably at least 99% of acompound of the invention by weight of the isolate.

The phrase “pharmaceutically acceptable salt(s),” as used hereinincludes but is not limited to salts of acidic or basic groups that maybe present in compounds used in the present compositions. Compoundsincluded in the present compositions that are basic in nature arecapable of forming a wide variety of salts with various inorganic andorganic acids. The acids that may be used to prepare pharmaceuticallyacceptable acid addition salts of such basic compounds are those thatform non-toxic acid addition salts, i.e., salts containingpharmacologically acceptable anions including, but not limited to,sulfuric, citric, maleic, acetic, oxalic, hydrochloride, hydrobromide,hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate,isonicotinate, acetate, lactate, salicylate, citrate, acid citrate,tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucaronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Compounds includedin the present compositions that include an amino moiety may formpharmaceutically acceptable salts with various amino acids, in additionto the acids mentioned above. Compounds, included in the presentcompositions, that are acidic in nature are capable of forming basesalts with various pharmacologically acceptable cations. Examples ofsuch salts include alkali metal or alkaline earth metal salts and,particularly, calcium, magnesium, sodium lithium, zinc, potassium, andiron salts.

As used herein and unless otherwise indicated, the term“pharmaceutically acceptable prodrug” means a derivative of a compoundthat can hydrolyze, oxidize, or otherwise react under biologicalconditions (in vitro or in vivo) to provide the compound. Examples ofprodrugs include, but are not limited to, compounds that comprisebiohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzableesters, biohydrolyzable carbamates, biohydrolyzable carbonates,biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Otherexamples of prodrugs include compounds that comprise oligonucleotides,peptides, lipids, aliphatic and aromatic groups, or NO, NO₂, ONO, andONO₂ moieties. Prodrugs can typically be prepared using well knownmethods, such as those described in Burger's Medicinal Chemistry andDrug Discovery, pp. 172, 178, 949, 982 (Manfred E. Wolff ed., 5th ed.1995), and Design of Prodrugs (H. Bundgaard ed., Elselvier, New York1985).

As used herein and unless otherwise indicated, the terms“biohydrolyzable amide,” “biohydrolyzable ester,” “biohydrolyzablecarbamate,” “biohydrolyzable carbonate,” “biohydrolyzable ureide,”“biohydrolyzable phosphate” mean an amide, ester, carbamate, carbonate,ureide, or phosphate, respectively, of a compound that either: 1) doesnot interfere with the biological activity of the compound but canconfer upon that compound advantageous properties in vivo, such asuptake, duration of action, or onset of action; or 2) is biologicallyinactive but is converted in vivo to the biologically active compound.Examples of biohydrolyzable esters include, but are not limited to,lower alkyl esters, lower acyloxyalkyl esters (such as acetoxylmethyl,acetoxyethyl, aminocarbonyloxy-methyl, pivaloyloxymethyl, andpivaloyloxyethyl esters), lactonyl esters (such as phthalidyl andthiophthalidyl esters), lower alkoxyacyloxyalkyl esters (such asmethoxycarbonyloxy-methyl, ethoxycarbonyloxy-ethyl andisopropoxycarbonyloxyethyl esters), alkoxyalkyl esters, choline esters,and acylamino alkyl esters (such as acetamidomethyl esters). Examples ofbiohydrolyzable amides include, but are not limited to, lower alkylamides, a amino acid amides, alkoxyacyl amides, andalkylaminoalkyl-carbonyl amides. Examples of biohydrolyzable carbamatesinclude, but are not limited to, lower alkylamines, substitutedethylenediamines, aminoacids, hydroxyalkylamines, heterocyclic andheteroaromatic amines, and polyether amines.

As used herein and unless otherwise indicated, the term “phenyl” means—C₆H₅. A phenyl group can be unsubstituted or substituted with one ortwo suitable substituents.

As used herein and unless otherwise indicated, the terms “substituted”and “a suitable substituent” means a group that does not nullify thesynthetic or pharmaceutical utility of the compounds of the invention orthe intermediates useful for preparing them. Examples of substitutedgroups or suitable substituents include, but are not limited to:(C₁-C₈)alkyl; (C₁-C₈)alkenyl; (C₁-C₈)alkynyl; (C₆)aryl;(C₃-C₅)heteroaryl; (C₃-C₇)cycloalkyl; (C₁-C₈)alkoxy; (C₆)aryloxy; —CN;—OH; SH, oxo; halo, —NO₂, —CO₂H; —NH₂; —NHOH, —NH((C₁-C₈)alkyl);—N((C₁-C₈)alkyl)₂; —NH((C₆)aryl); —NHO((C₁-C₈)alkyl);—N(O(C₁-C₈)alkyl)₂; —NH(O(C₆)aryl); —S((C₁-C₈)alkyl); —S((C₁-C₈)alkyl)₂;—S((C₆)aryl); (═O); C(S), —N((C₆)aryl)₂; —CHO; —C(O)((C₁-C₈)alkyl);—C(O)((C₆)aryl); —CO₂((C₁-C₈)alkyl); and —CO₂((C₆)aryl),—C(S)((C₁-C₈)alkyl); —C(S)((C₆)aryl); —SO₂((C₁-C₈)alkyl);—SO₂((C₆)aryl), and —SO₃H, —C(S)O((C₁-C₈)alkyl); —C(S)(O)((C₆)aryl). Incertain illustrative embodiments, the substituents can be one or morethan one suitable groups, such as, but not limited to, —F, —Cl, —Br, —I,—OH, azido, —SH, alkyl, aryl, heteroalky, alkyoxyl, alkylthiol, amino,hydroxylamino, N-alkylamino, —N,N-dialkylamino, —N,N-dimethylamino,acyl, alkyloxycarbonyl, sulfonyl, urea, —NO₂, triazolyl. One of skill inart can readily choose a suitable substituent based on the stability andpharmacological and synthetic activity of the compound of the invention.

As used herein and unless otherwise indicated, the phrase“therapeutically effective amount” of a composition of the invention ismeasured by the therapeutic effectiveness of a compound of theinvention, wherein at least one adverse effect of a disorder isameliorated or alleviated.

The terms “treating or preventing” are intended to include preventing,eradicating, or inhibiting the resulting increase of undesiredphysiological activity associated with a disorder, for example, in thecontext of the therapeutic or prophylactic methods of the invention. Inanother embodiment, the term treating or preventing includesantagonistic effects, e.g., diminishment of the activity or productionof mediators of a disorder.

As used herein, the term “proliferative disease”, “hyperproliferativedisorder”, “disease caused by uncontrolled cell growth” and the likeincludes cancer and other diseases such as neoplasias and hyperplasias.Cellular proliferative diseases include, for example, rheumatoidarthritis, inflammatory bowel disease, osteoarthritis, leiomyomas,adenomas, lipomas, hemangiomas, fibromas, vascular occlusion,restenosis, artherosclerosis, a preneoplastic lesion, carcinoma in situ,oral hairy leukoplakia, or psoriasis. Cancer and tumors are particularexamples of proliferative disease. In accordance with one or moreembodiments, the term cancer can include, for example cancers of thelung, liver, pancreas, prostate, breast, kidney, and central nervoussystem including the brain (for example, glioblastoma, medulloblastoma,and pontine tumors), related tumors. In accordance with one or moreembodiments, the cancers treated by the present invention includecancers which are resistant to hypoxia, or chemotherapy, such as, forexample, tamoxifen or taxol resistant cancers.

Exemplary Compounds Useful for Practicing the Invention

As set forth herein, the compounds useful in practicing the inventioninclude, but are not limited to, compounds as described in Formula I-Vset forth herein, as well as specific compounds disclosed elsewhere inthis specification, and compositions and formulations containing thosecompounds or a pharmaceutically acceptable salt or prodrug thereof.These compounds are useful for treatment and identification methods ofthe invention, for example for treating, preventing or identifying adisease or disorder susceptible to treatment by downregulation of DDX3expression including, but not limited to, conditions caused byuncontrolled cell growth, hyperproliferation of cells, tumor growth, andcancers, for example, pancreatic cancer, liver cancer, breast cancer,lung cancer, prostate cancer, brain cancer including glioblastoma,medulloblastoma, and pontine tumors, kidney cancer, leukemia, or asarcoma, which comprises administering to a mammal in need of suchtreatment or prevention a therapeutically or prophylactically effectiveamount of a composition comprising a compound of Formula I-V, or apharmaceutically acceptable salt or prodrug thereof, and apharmaceutically acceptable vehicle. In certain embodiments, acomposition or formulation comprising a compound of Formula I-V isuseful in treating or preventing conditions caused by uncontrolled cellgrowth. In certain embodiments, a composition or formulation comprisinga compound of Formula I-V is useful in killing abnormal or cancerouscells while simultaneously not affecting healthy or normal cells. Incertain embodiments, a composition or formulation comprising a compoundof Formula I-V act as cytotoxic agents. In certain embodiments, acomposition or formulation comprising a compound of Formula I-V act asapoptotic agents.

The invention encompasses methods of treating or preventing diseases anddisorders described herein by administering a composition or formulationcomprising a compound of Formulas I-V or a pharmaceutically acceptablesalt or prodrug thereof.

As described herein, the compositions that are useful in the methods ofthe invention encompass compounds of Formulas I-V.

In one embodiment, the invention encompasses compounds and compositionsand formulations for treating or preventing diseases or disorderscomprising a compound of Formula (I):

or pharmaceutically acceptable salts and prodrugs thereof,wherein:

-   R, R′, and R″ are each independently a hydrogen, hydroxyl;    substituted or unsubstituted: cyclic or acyclic alkyl group, cyclic    or acyclic alkenyl group, cyclic or acyclic alkynyl group, aryl    group, alkylaryl group, arylalkyl group, benzyl group, cyclic and    acyclic heteroalkyl group, heteroaryl group; —C(O)R³; —C(S)R³;    —S(O)R³; —S(O)₂R³; —C(O)NR³R⁴; —C(S)NR³R⁴; —C(S)YR³; —C(O)YR³;    -β-D-ribosyl; -α-D-ribosyl; -β-L-ribosyl; -α-L-ribosyl;    2′-deoxy-β-D-ribosyl; 2′-deoxy-β-L-ribosyl; 2′-deoxy-α-D-ribosyl;    2′-deoxy-α-L-ribosyl; or ribose or deoxyribose sugars substituted    with one or more halogens;-   R, R′, and R″ can also form a ring with one or more C, S, O, N atoms    such that, for example, R and R′ together include:

-   R⁷ is a hydrogen; hydroxyl; substituted and unsubstituted: cyclic    and acyclic alkyl group, group, alkenyl group, alkynyl group, aryl    group, aryloxy group, alkylary group, aryalkyl group, heteroaryl    group, heterocycloalkyl group; —C(O)alkyl; —C(O)alkenyl;    —C(O)alkynyl;    -   —C(O)aryl; —C(O)benzyl; —C(O)NR³R⁴; —C(S)alkyl; —C(S)alkenyl;        —C(S)alkynyl;    -   —C(S)aryl; —C(S)benzyl; —C(S)NR³R⁴; —C(O)NR³R⁴; —C(S)YR³;        —C(O)YR³;    -   wherein-   - - - - - Q is ═O, ═NH, or ═S;-   Y is O or S;-   Z is CH, N, P, or C;-   — is a single bond or double bond; wherein if — is a double bond, R²    or R⁷ is independently O, S, or NH;-   n is 1, 2, 3, or 4;-   R³ and R⁴ are independently a hydrogen; hydroxyl; substituted or    unsubstituted: cyclic or acyclic alkyl group, cyclic or acyclic    alkenyl group, cyclic or acyclic alkynyl group, aryl group, alkylary    group, aryalkyl group, heteroaryl group, heterocycloalkyl group; and-   r, r′, and r″ are each independently an integer from 1 to 3.

In certain illustrative embodiments, R, R′, and R″ are eachindependently are a substituted benzyl, alkyl, aryl, cycloalkyl,heteroaryl, or heterocycloalkyl with one or more substituents, such as,but not limited to, —H, —F, —Cl, —Br, —I, —OH, azido, —SH, alkyl, aryl,heteroalky, alkyoxyl, alkylthiol, amino, hydroxylamino, N-alkylamino,—N,N-dialkylamino, —N,N-dimethylamino, acyl, alkyloxycarbonyl, sulfonyl,urea, —NO₂, triazolyl.

In certain illustrative embodiments, the compounds of the invention donot include compounds where both R, R′, and R″ are all hydrogen.

In certain illustrative embodiments, the substituents can be one or morethan one suitable groups, such as, but not limited to, —F, —Cl, —Br, —I,—OH, azido, —SH, alkyl, aryl, heteroalky, alkyoxyl, alkylthiol, amino,hydroxylamino, N-alkylamino, —N,N-dialkylamino, —N,N-dimethylamino,acyl, alkyloxycarbonyl, sulfonyl, urea, —NO₂, triazolyl.

In one embodiment, R is hydrogen. In another embodiment, R is an alkylgroup. In another embodiment, R is an alkoxy group. In anotherembodiment, R is an alkylalkoxy group. In another embodiment, R is analkenyl group. In another embodiment, R is alkynyl group. In anotherembodiment, R is an aryl group. In another embodiment, R is aryloxygroup. In another embodiment, R is benzyl group. In another embodiment,R is heteroaryl group. In another embodiment, R is heterocycloalkylgroup. In another embodiment, R is a cycloalkyl group. In anotherembodiment, R is a benzyl group.

In certain illustrative embodiments, R is a benzyl group substitutedwith one or more groups or atoms selected from F, Cl, Br, I, OH, azido,SH, alkyl, aryl, heteroalkyl, alkyloxyl, alkylthiol, amino,hydroxylamino, N-alkylamino, N,N-dialkylamino, N,N-dimethylamino, acyl,alkyloxycarbonyl, sulfonyl, urea, NO₂, and triazolyl.

In one embodiment, R′ is hydrogen. In another embodiment, R′ is an alkylgroup. In another embodiment, R′ is an alkoxy group. In anotherembodiment, R′ is an alkylalkoxy group. In another embodiment, R′ is analkenyl group. In another embodiment, R′ is alkynyl group. In anotherembodiment, R′ is an aryl group. In another embodiment, R′ is aryloxygroup. In another embodiment, R′ is benzyl group. In another embodiment,R′ is heteroaryl group. In another embodiment, R′ is heterocycloalkylgroup. In another embodiment, R′ is a cycloalkyl group. In anotherembodiment, R′ is a benzyl group.

In certain illustrative embodiments, R′ is a benzyl group substitutedwith one or more groups or atoms selected from F, Cl, Br, I, OH, azido,SH, alkyl, aryl, heteroalkyl, alkyloxyl, alkylthiol, amino,hydroxylamino, N-alkylamino, N,N-dialkylamino, N,N-dimethylamino, acyl,alkyloxycarbonyl, sulfonyl, urea, NO₂, or triazolyl.

In another embodiment, R″ is hydrogen. In another embodiment, R″ is analkyl group. In another embodiment, R″ is an alkoxy group. In anotherembodiment, R″ is an alkylalkoxy group. In another embodiment, R″ is analkenyl group. In another embodiment, R″ is alkynyl group. In anotherembodiment, R″ is an aryl group. In another embodiment, R″ is aryloxygroup. In another embodiment, R″ is benzyl group. In another embodiment,R″ is heteroaryl group. In another embodiment, R″ is heterocycloalkylgroup. In another embodiment, R″ is a cycloalkyl group. In anotherembodiment, R″ is a benzyl group.

In certain illustrative embodiments, R″ is a benzyl group substitutedwith one or more groups or atoms selected from F, Cl, Br, I, OH, azido,SH, alkyl, aryl, heteroalkyl, alkyloxyl, alkylthiol, amino,hydroxylamino, N-alkylamino, N,N-dialkylamino, N,N-dimethylamino, acyl,alkyloxycarbonyl, sulfonyl, urea, NO₂, or triazolyl.

In another embodiment, the invention encompasses compositions andformulations for treating or preventing diseases or disorders comprisinga compound of Formula (II):

or pharmaceutically acceptable salts and prodrugs thereof,wherein

-   R¹, R², R³, R⁴, and R⁵ are each independently a hydrogen; hydroxyl;    substituted or unsubstituted: cyclic or acyclic alkyl group, cyclic    or acyclic alkenyl group, cyclic or acyclic alkynyl group, aryl    group, alkylaryl group, arylalkyl group, benzyl group, cyclic or    acyclic heteroalkyl group, heteroaryl group; —C(O)R⁶; —C(S)R⁶;    —S(O)R⁶; —S(O)₂R⁶; —C(O)NR⁶R⁸; —C(S)NR⁶R⁸; C(S)YR⁶; —C(O)YR⁶;    -β-D-ribosyl; -α-D-ribosyl; -β-L-ribosyl; -α-L-ribosyl;    2′-deoxy-β-D-ribosyl; 2′-deoxy-β-L-ribosyl; 2′-deoxy-α-D-ribosyl;    2′-deoxy-α-L-ribosyl; or ribose or deoxyribose sugars substituted    with one or more halogens;-   R¹ and R³ or R² and R⁴ can also form a ring with one or more C, S,    O, N atoms such that R¹ and R³ or R² and R⁴ together include

-   R⁷ is a hydrogen; hydroxyl; substituted and unsubstituted: cyclic or    acyclic alkyl group, group, alkenyl group, alkynyl group, aryl    group, aryloxy group, alkylary group, aryalkyl group, heteroaryl    group, heterocycloalkyl group; —C(O)alkyl; —C(O)alkenyl;    —C(O)alkynyl; —C(O)aryl; —C(O)benzyl; —C(O)NR⁶R⁸; —C(S)alkyl;    —C(S)alkenyl; —C(S)alkynyl; —C(S)aryl; —C(S)benzyl; —C(S)NR⁶R⁸;    —C(O)YR⁶; or —C(S)YR⁶ wherein-   Y is O or S;-   Z is CH, N, P, or C;-   — is a single bond or double bond; wherein if — is a double bond, R²    or R⁷ is independently O, S, or NH;-   R⁶ and R⁸ are independently a hydrogen; hydroxyl; substituted or    unsubstituted: cyclic or acyclic alkyl group, cyclic or acyclic    alkenyl group, cyclic or acyclic alkynyl group, aryl group, alkylary    group, aryalkyl group, heteroaryl group, heterocycloalkyl group; and-   n is 1, 2, 3, or 4.

In certain illustrative embodiments, R¹, R², R³, R⁴, and R⁵ are eachindependently a substituted benzyl, alkyl, aryl, cycloalkyl, heteroaryl,or heterocycloalkyl with one or more substituents, such as, but notlimited to, —H, —F, —Cl, —Br, —I, —OH, azido, —SH, alkyl, aryl,heteroalky, alkyoxyl, alkylthiol, amino, hydroxylamino, N-alkylamino,—N,N-dialkylamino, —N,N-dimethylamino, acyl, alkyloxycarbonyl, sulfonyl,urea, —NO₂, triazolyl.

In one embodiment, R¹ is hydrogen. In another embodiment, R¹ is an alkylgroup. In another embodiment, R¹ is an alkoxy group. In anotherembodiment, R¹ is an alkylalkoxy group. In another embodiment, R¹ is analkenyl group. In another embodiment, R¹ is alkynyl group. In anotherembodiment, R¹ is an aryl group. In another embodiment, R¹ is aryloxygroup. In another embodiment, R¹ is benzyl group. In another embodiment,R¹ is heteroaryl group. In another embodiment, R¹ is heterocycloalkylgroup. In another embodiment, R¹ is a cycloalkyl group. In anotherembodiment, R¹ is a benzyl group.

In certain illustrative embodiments, R¹ is a benzyl group substitutedwith one or more groups or atoms selected from F, Cl, Br, I, OH, azido,SH, alkyl, aryl, heteroalkyl, alkyloxyl, alkylthiol, amino,hydroxylamino, N-alkylamino, N,N-dialkylamino, N,N-dimethylamino, acyl,alkyloxycarbonyl, sulfonyl, urea, NO₂, or triazolyl.

In one embodiment, R² is hydrogen. In another embodiment, R² is an alkylgroup. In another embodiment, R² is an alkoxy group. In anotherembodiment, R² is an alkylalkoxy group. In another embodiment, R² is analkenyl group. In another embodiment, R² is alkynyl group. In anotherembodiment, R² is an aryl group. In another embodiment, R² is aryloxygroup. In another embodiment, R² is benzyl group. In another embodiment,R² is heteroaryl group. In another embodiment, R² is heterocycloalkylgroup. In another embodiment, R² is a cycloalkyl group. In anotherembodiment, R² is a benzyl group.

In certain illustrative embodiments, R² is a benzyl group substitutedwith one or more groups or atoms selected from F, Cl, Br, I, OH, azido,SH, alkyl, aryl, heteroalkyl, alkyloxyl, alkylthiol, amino,hydroxylamino, N-alkylamino, N,N-dialkylamino, N,N-dimethylamino, acyl,alkyloxycarbonyl, sulfonyl, urea, NO₂, or triazolyl.

In one embodiment, R³ is hydrogen. In another embodiment, R³ is an alkylgroup. In another embodiment, R³ is an alkoxy group. In anotherembodiment, R³ is an alkylalkoxy group. In another embodiment, R³ is analkenyl group. In another embodiment, R³ is alkynyl group. In anotherembodiment, R³ is an aryl group. In another embodiment, R³ is aryloxygroup. In another embodiment, R³ is benzyl group. In another embodiment,R³ is heteroaryl group. In another embodiment, R³ is heterocycloalkylgroup. In another embodiment, R³ is a cycloalkyl group. In anotherembodiment, R³ is a benzyl group.

In certain illustrative embodiments, R³ is a benzyl group substitutedwith one or more groups or atoms selected from F, Cl, Br, I, OH, azido,SH, alkyl, aryl, heteroalkyl, alkyloxyl, alkylthiol, amino,hydroxylamino, N-alkylamino, N,N-dialkylamino, N,N-dimethylamino, acyl,alkyloxycarbonyl, sulfonyl, urea, NO₂, or triazolyl.

In one embodiment, R⁴ is hydrogen. In another embodiment, R⁴ is an alkylgroup. In another embodiment, R⁴ is an alkoxy group. In anotherembodiment, R⁴ is an alkylalkoxy group. In another embodiment, R⁴ is analkenyl group. In another embodiment, R⁴ is alkynyl group. In anotherembodiment, R⁴ is an aryl group. In another embodiment, R⁴ is aryloxygroup. In another embodiment, R⁴ is benzyl group. In another embodiment,R⁴ is heteroaryl group. In another embodiment, R⁴ is heterocycloalkylgroup. In another embodiment, R⁴ is a cycloalkyl group. In anotherembodiment, R⁴ is a benzyl group.

In certain illustrative embodiments, R⁴ is a benzyl group substitutedwith one or more groups or atoms selected from F, Cl, Br, I, OH, azido,SH, alkyl, aryl, heteroalkyl, alkyloxyl, alkylthiol, amino,hydroxylamino, N-alkylamino, N,N-dialkylamino, N,N-dimethylamino, acyl,alkyloxycarbonyl, sulfonyl, urea, NO₂, or triazolyl. In one embodiment,R⁵ is hydrogen. In another embodiment, R⁵ is an alkyl group. In anotherembodiment, R⁵ is an alkoxy group. In another embodiment, R⁵ is analkylalkoxy group. In another embodiment, R⁵ is an alkenyl group. Inanother embodiment, R⁵ is alkynyl group. In another embodiment, R⁵ is anaryl group. In another embodiment, R⁵ is aryloxy group. In anotherembodiment, R⁵ is benzyl group. In another embodiment, R⁵ is heteroarylgroup. In another embodiment, R⁵ is heterocycloalkyl group. In anotherembodiment, R⁵ is a cycloalkyl group. In another embodiment, R⁵ is abenzyl group.

In certain illustrative embodiments, R⁵ is a benzyl group substitutedwith one or groups or atoms selected from F, Cl, Br, I, OH, azido, SH,alkyl, aryl, heteroalkyl, alkyloxyl, alkylthiol, amino, hydroxylamino,N-alkylamino, N,N-dialkylamino, N,N-dimethylamino, acyl,alkyloxycarbonyl, sulfonyl, urea, NO₂, or triazolyl.

In another embodiment, the invention encompasses compositions andformulations for treating or preventing diseases or disorders comprisinga compound of Formula (III):

or pharmaceutically acceptable salts and prodrugs thereof,wherein

-   R¹, R³, R⁴, and R⁵ are each independently a hydrogen; hydroxyl;    substituted or unsubstituted: cyclic or acyclic alkyl group, cyclic    or acyclic alkenyl group, cyclic or acyclic alkynyl group, aryl    group, alkylaryl group, arylalkyl group, benzyl group, cyclic and    acyclic heteroalkyl group, heteroaryl group; —C(O)R⁶; —C(S)R⁶;    —S(O)R⁶; —S(O)₂R⁶; —C(O)NR⁶R⁷; —C(S)NR⁶R⁷; C(S)YR⁶; —C(O)YR⁶;    -β-D-ribosyl; -α-D-ribosyl; -β-L-ribosyl; -α-L-ribosyl;    2′-deoxy-β-D-ribosyl; 2′-deoxy-β-L-ribosyl; 2′-deoxy-α-D-ribosyl;    2′-deoxy-α-L-ribosyl; or ribose or deoxyribose sugars substituted    with one or more halogens;-   R¹ and R³ can also form a ring with one or more C, S, O, N atoms    such that R¹ and R³ together include

-   R⁷ is a hydrogen; hydroxyl; substituted and unsubstituted: cyclic or    acyclic alkyl group, group, alkenyl group, alkynyl group, aryl    group, aryloxy group, alkylary group, aryalkyl group, heteroaryl    group, heterocycloalkyl group; —C(O)alkyl; —C(O)alkenyl;    —C(O)alkynyl;    -   —C(O)aryl; —C(O)benzyl; —C(O)NR³R⁴; —C(S)alkyl; —C(S)alkenyl;        —C(S)alkynyl;    -   —C(S)aryl; —C(S)benzyl; —C(S)NR³R⁴; —C(O)NR³R⁴; —C(S)YR³;        —C(O)YR³; wherein-   Y is O or S;-   Z is CH, N, P, or C;-   — is a single bond or double bond; wherein if — is a double bond, R²    or R⁷ is independently O, S, or NH;-   R⁶ and R⁸ are independently a hydrogen; hydroxyl; substituted or    unsubstituted: cyclic or acyclic alkyl group, cyclic or acyclic    alkenyl group, cyclic or acyclic alkynyl group, aryl group, alkylary    group, aryalkyl group, heteroaryl group, heterocycloalkyl group; and-   n is 1, 2, 3, or 4.

In certain illustrative embodiments, R¹, R³, R⁴, and R⁵ are not allhydrogen.

In certain illustrative embodiments, R¹, R³, R⁴, and R⁵ are eachindependently are a substituted benzyl, alkyl, aryl, cycloalkyl,heteroaryl, or heterocycloalkyl with one ore more substituents, such as,but not limited to, —H, —F, —Cl, —Br, —I, —OH, azido, —SH, alkyl, aryl,heteroalky, alkyoxyl, alkylthiol, amino, hydroxylamino, N-alkylamino,—N,N-dialkylamino, —N,N-dimethylamino, acyl, alkyloxycarbonyl, sulfonyl,urea, —NO₂, triazolyl.

In one embodiment, R¹ is hydrogen. In another embodiment, R¹ is an alkylgroup. In another embodiment, R¹ is an alkoxy group. In anotherembodiment, R¹ is an alkylalkoxy group. In another embodiment, R¹ is analkenyl group. In another embodiment, R¹ is alkynyl group. In anotherembodiment, R¹ is an aryl group. In another embodiment, R¹ is aryloxygroup. In another embodiment, R¹ is benzyl group. In another embodiment,R¹ is heteroaryl group. In another embodiment, R¹ is heterocycloalkylgroup. In another embodiment, R¹ is a cycloalkyl group. In anotherembodiment, R¹ is a benzyl group.

In certain illustrative embodiments, R¹ is a benzyl group substitutedwith one or more groups or atoms selected from F, Cl, Br, I, OH, azido,SH, alkyl, aryl, heteroalkyl, alkyloxyl, alkylthiol, amino,hydroxylamino, N-alkylamino, N,N-dialkylamino, N,N-dimethylamino, acyl,alkyloxycarbonyl, sulfonyl, urea, NO₂, or triazolyl. In one embodiment,R³ is hydrogen. In another embodiment, R³ is an alkyl group. In anotherembodiment, R³ is an alkoxy group. In another embodiment, R³ is analkylalkoxy group. In another embodiment, R³ is an alkenyl group. Inanother embodiment, R³ is alkynyl group. In another embodiment, R³ is anaryl group. In another embodiment, R³ is aryloxy group. In anotherembodiment, R³ is benzyl group. In another embodiment, R³ is heteroarylgroup. In another embodiment, R³ is heterocycloalkyl group. In anotherembodiment, R³ is a cycloalkyl group. In another embodiment, R³ is abenzyl group.

In certain illustrative embodiments, R³ is a benzyl group substitutedwith one or more groups or atoms selected from F, Cl, Br, I, OH, azido,SH, alkyl, aryl, heteroalkyl, alkyloxyl, alkylthiol, amino,hydroxylamino, N-alkylamino, N,N-dialkylamino, N,N-dimethylamino, acyl,alkyloxycarbonyl, sulfonyl, urea, NO₂, or triazolyl.

In one embodiment, R⁴ is hydrogen. In another embodiment, R⁴ is an alkylgroup. In another embodiment, R⁴ is an alkoxy group. In anotherembodiment, R⁴ is an alkylalkoxy group. In another embodiment, R⁴ is analkenyl group. In another embodiment, R⁴ is alkynyl group. In anotherembodiment, R⁴ is an aryl group. In another embodiment, R⁴ is aryloxygroup. In another embodiment, R⁴ is benzyl group. In another embodiment,R⁴ is heteroaryl group. In another embodiment, R⁴ is heterocycloalkylgroup. In another embodiment, R⁴ is a cycloalkyl group. In anotherembodiment, R⁴ is a benzyl group.

In certain illustrative embodiments, R⁴ is a benzyl group substitutedwith one or more groups or atoms selected from F, Cl, Br, I, OH, azido,SH, alkyl, aryl, heteroalkyl, alkyloxyl, alkylthiol, amino,hydroxylamino, N-alkylamino, N,N-dialkylamino, N,N-dimethylamino, acyl,alkyloxycarbonyl, sulfonyl, urea, NO₂, or triazolyl.

In one embodiment, R⁵ is hydrogen. In another embodiment, R⁵ is an alkylgroup. In another embodiment, R⁵ is an alkoxy group. In anotherembodiment, R⁵ is an alkylalkoxy group. In another embodiment, R⁵ is analkenyl group. In another embodiment, R⁵ is alkynyl group. In anotherembodiment, R⁵ is an aryl group. In another embodiment, R⁵ is aryloxygroup. In another embodiment, R⁵ is benzyl group. In another embodiment,R⁵ is heteroaryl group. In another embodiment, R⁵ is heterocycloalkylgroup. In another embodiment, R⁵ is a cycloalkyl group. In anotherembodiment, R⁵ is a benzyl group.

In certain illustrative embodiments, R⁵ is a benzyl group substitutedwith one or more groups or atoms selected from F, Cl, Br, I, OH, azido,SH, alkyl, aryl, heteroalkyl, alkyloxyl, alkylthiol, amino,hydroxylamino, N-alkylamino, N,N-dialkylamino, N,N-dimethylamino, acyl,alkyloxycarbonyl, sulfonyl, urea, NO₂, or triazolyl.

In another embodiment, the invention encompasses compositions andformulations for treating or preventing diseases or disorders comprisinga compound of Formula (IV):

or pharmaceutically acceptable salts and prodrugs thereof,wherein

-   R³ and R⁴ are each independently a hydrogen; hydroxyl; substituted    or unsubstituted: cyclic or acyclic alkyl group, cyclic or acyclic    alkenyl group, cyclic a or nd acyclic alkynyl group, aryl group,    alkylaryl group, arylalkyl group, benzyl group, cyclic or acyclic    heteroalkyl group, heteroaryl group; —C(O)R⁵; —C(S)R⁵; —S(O)R⁵;    —S(O)₂R⁵; —C(O)NR⁵R⁶; —C(S)NR⁵R⁶; —C(O)YR⁵; —C(S)YR⁵; -β-D-ribosyl;    -α-D-ribosyl; -β-L-ribosyl; -α-L-ribosyl; 2′-deoxy-β-D-ribosyl;    2′-deoxy-β-L-ribosyl; 2′-deoxy-α-D-ribosyl; 2′-deoxy-α-L-ribosyl; or    ribose or deoxyribose sugars substituted with one or more halogens;-   R⁵ and R⁶ are independently a hydrogen; hydroxyl; substituted or    unsubstituted: cyclic or acyclic alkyl group, cyclic or acyclic    alkenyl group, cyclic or acyclic alkynyl group, aryl group, alkylary    group, aryalkyl group, heteroaryl group, heterocycloalkyl group; and-   Y is O or S.

In certain illustrative embodiments, R³ and R⁴ are both not hydrogen.

In certain illustrative embodiments, R³ and R⁴ are each independentlyare a substituted benzyl, alkyl, aryl, cycloalkyl, heteroaryl, orheterocycloalkyl with one or more substituents, such as, but not limitedto, —H, —F, —Cl, —Br, —I, —OH, azido, —SH, alkyl, aryl, heteroalky,alkyoxyl, alkylthiol, amino, hydroxylamino, N-alkylamino,—N,N-dimethylamino, acyl, alkyloxycarbonyl, sulfonyl, urea, —NO₂,triazolyl.

In one embodiment, R³ is hydrogen. In another embodiment, R³ is an alkylgroup. In another embodiment, R³ is an alkoxy group. In anotherembodiment, R³ is an alkylalkoxy group. In another embodiment, R³ is analkenyl group. In another embodiment, R³ is alkynyl group. In anotherembodiment, R³ is an aryl group. In another embodiment, R³ is aryloxygroup. In another embodiment, R³ is benzyl group. In another embodiment,R³ is heteroaryl group. In another embodiment, R³ is heterocycloalkylgroup. In another embodiment, R³ is a cycloalkyl group. In anotherembodiment, R³ is a benzyl group.

In certain illustrative embodiments, R³ is a benzyl group substitutedwith one or more groups or atoms selected from F, Cl, Br, I, OH, azido,SH, alkyl, aryl, heteroalkyl, alkyloxyl, alkylthiol, amino,hydroxylamino, N-alkylamino, N,N-dialkylamino, N,N-dimethylamino, acyl,alkyloxycarbonyl, sulfonyl, urea, NO₂, or triazolyl.

In one embodiment, R⁴ is hydrogen. In another embodiment, R⁴ is an alkylgroup. In another embodiment, R⁴ is an alkoxy group. In anotherembodiment, R⁴ is an alkylalkoxy group. In another embodiment, R⁴ is analkenyl group. In another embodiment, R⁴ is alkynyl group. In anotherembodiment, R⁴ is an aryl group. In another embodiment, R⁴ is aryloxygroup. In another embodiment, R⁴ is benzyl group. In another embodiment,R⁴ is heteroaryl group. In another embodiment, R⁴ is heterocycloalkylgroup. In another embodiment, R⁴ is a cycloalkyl group. In anotherembodiment, R⁴ is a benzyl group.

In certain illustrative embodiments, R⁴ is a benzyl group substitutedwith one or more groups or atoms selected from F, Cl, Br, I, OH, azido,SH, alkyl, aryl, heteroalkyl, alkyloxyl, alkylthiol, amino,hydroxylamino, N-alkylamino, N,N-dialkylamino, N,N-dimethylamino, acyl,alkyloxycarbonyl, sulfonyl, urea, NO₂, or triazolyl.

In another embodiment, the invention encompasses compositions andformulations for treating or preventing diseases or disorders comprisinga compound of Formula (V):

or pharmaceutically acceptable salts and prodrugs thereof,wherein

-   R³, R⁴, and R⁵ are each independently a hydrogen; hydroxyl;    substituted or unsubstituted: cyclic or acyclic alkyl group, cyclic    or acyclic alkenyl group, cyclic or acyclic alkynyl group, aryl    group, alkylaryl group, arylalkyl group, benzyl group, cyclic or    acyclic heteroalkyl group, heteroaryl group; —C(O)R⁶; —C(S)R⁶;    —S(O)R⁶; —S(O)₂R⁶; —C(O)NR⁶R⁷; —C(S)NR⁶R⁷; C(S)YR⁶; —C(O)YR⁶;    -β-D-ribosyl; -α-D-ribosyl; -β-L-ribosyl; -α-L-ribosyl;    2′-deoxy-β-D-ribosyl; 2′-deoxy-β-L-ribosyl; 2′-deoxy-α-D-ribosyl;    2′-deoxy-α-L-ribosyl; or ribose or deoxyribose sugars substituted    with one or more halogens;-   R⁵ and R⁶ are independently a hydrogen; hydroxyl; substituted or    unsubstituted: cyclic or acyclic alkyl group, cyclic or acyclic    alkenyl group, cyclic or acyclic alkynyl group, aryl group, alkylary    group, aryalkyl group, heteroaryl group, heterocycloalkyl group; and-   Y is O or S.

In certain illustrative embodiments, R³, R⁴, and R⁵ are not eachhydrogen.

In certain illustrative embodiments, R³, R⁴, and R⁵ are eachindependently are a substituted benzyl, alkyl, aryl, cycloalkyl,heteroaryl, or heterocycloalkyl with one ore more substituents, such as,but not limited to, —H, —F, —Cl, —Br, —I, —OH, azido, —SH, alkyl, aryl,heteroalky, alkyoxyl, alkylthiol, amino, hydroxylamino, N-alkylamino,—N,N-dialkylamino, —N,N-dimethylamino, acyl, alkyloxycarbonyl, sulfonyl,urea, —NO₂, triazolyl.

In one embodiment, R³ is hydrogen. In another embodiment, R³ is an alkylgroup. In another embodiment, R³ is an alkoxy group. In anotherembodiment, R³ is an alkylalkoxy group. In another embodiment, R³ is analkenyl group. In another embodiment, R³ is alkynyl group. In anotherembodiment, R³ is an aryl group. In another embodiment, R³ is aryloxygroup. In another embodiment, R³ is benzyl group. In another embodiment,R³ is heteroaryl group. In another embodiment, R³ is heterocycloalkylgroup. In another embodiment, R³ is a cycloalkyl group. In anotherembodiment, R³ is a benzyl group.

In certain illustrative embodiments, R³ is a benzyl group substitutedwith one or more groups or atoms selected from F, Cl, Br, I, OH, azido,SH, alkyl, aryl, heteroalkyl, alkyloxyl, alkylthiol, amino,hydroxylamino, N-alkylamino, N,N-dialkylamino, N,N-dimethylamino, acyl,alkyloxycarbonyl, sulfonyl, urea, NO₂, or triazolyl.

In one embodiment, R⁴ is hydrogen. In another embodiment, R⁴ is an alkylgroup. In another embodiment, R⁴ is an alkoxy group. In anotherembodiment, R⁴ is an alkylalkoxy group. In another embodiment, R⁴ is analkenyl group. In another embodiment, R⁴ is alkynyl group. In anotherembodiment, R⁴ is an aryl group. In another embodiment, R⁴ is aryloxygroup. In another embodiment, R⁴ is benzyl group. In another embodiment,R⁴ is heteroaryl group. In another embodiment, R⁴ is heterocycloalkylgroup. In another embodiment, R⁴ is a cycloalkyl group. In anotherembodiment, R⁴ is a benzyl group.

In certain illustrative embodiments, R⁴ is a benzyl group substitutedwith one or more groups or atoms selected from F, Cl, Br, I, OH, azido,SH, alkyl, aryl, heteroalkyl, alkyloxyl, alkylthiol, amino,hydroxylamino, N-alkylamino, N,N-dialkylamino, N,N-dimethylamino, acyl,alkyloxycarbonyl, sulfonyl, urea, NO₂, or triazolyl.

In one embodiment, R⁵ is hydrogen. In another embodiment, R⁵ is an alkylgroup. In another embodiment, R⁵ is an alkoxy group. In anotherembodiment, R⁵ is an alkylalkoxy group. In another embodiment, R⁵ is analkenyl group. In another embodiment, R⁵ is alkynyl group. In anotherembodiment, R⁵ is an aryl group. In another embodiment, R⁵ is aryloxygroup. In another embodiment, R⁵ is benzyl group. In another embodiment,R⁵ is heteroaryl group. In another embodiment, R⁵ is heterocycloalkylgroup. In another embodiment, R⁵ is a cycloalkyl group. In anotherembodiment, R⁵ is a benzyl group.

In certain illustrative embodiments, R⁵ is a phenyl or a benzyl groupsubstituted with one or more groups or atoms selected from F, Cl, Br, I,OH, azido, SH, alkyl, aryl, heteroalkyl, alkyloxyl, alkylthiol, amino,hydroxylamino, N-alkylamino, N,N-dialkylamino, N,N-dimethylamino, acyl,alkyloxycarbonyl, sulfonyl, urea, NO₂, or triazolyl.

Illustrative examples of compounds that are encompassed by Formulas I-Vand that are useful in the methods of the invention include, but are notlimited to:

It will be understood that above compounds are illustrative only and notintended to limit the scope of the claims to only those compounds.

The compounds of the invention can be synthesized by organic chemistrytechniques known to those of ordinary skill in the art, for example, asdescribed in International Application No. PCT/US2009/005273. Anexemplary synthesis is generally described in Scheme 1 below.

The target 5:7:5 heterocyclic ring system was yet unknown. Scheme 2illustrates the first entry into such a ring system employing anillustrative representative example 3, containing a removablep-methoxybenzyl (PMB) group attached to each of the two imidazole rings.

Generally, an illustrative compound of the invention can be synthesizedby heating diaminomaleonitrile 4 with triethyl orthoformate in dioxaneyielded formimidate 5. (See, e.g., Sun, Z.; Hosmane, R. S. Synth.Commun. 2001, 31, 549). The reaction of 5 with p-methoxybenzylaminecatalyzed by aniline hydrochloride formed formimidine 6 (See, e.g.,Yahya-Zadeh, A.; Booth, B. L. Synth. Commun. 2001, 31, 3225), whichunderwent intramolecular cyclization in the presence of DBU to formimidazole derivative 7. (See, e.g., Yahyazadeh, A.; Sharifi, Z.Phosphorus, Sulfur, Silicon, Relat. Elem. 2006, 181, 1339). Thetreatment of latter with p-methoxybenzyl isocyanate resulted into amixture of urea 8 and 9. (See, e.g., Dias, A. M.; Cabral, I.; Proenca,M. F.; Booth, B. L. J. Org. Chem. 2002, 67, 5546). The completecoversion of 8 into 9 was achieved by treating the mixture with DBU inacetonitrile. (Id.). The reaction of isolated 9 with triethylorthoformate yielded the target heterocycle 3. In certain embodiments,the reported rearrangement of a compound such as 8 into an oxopurinesuch as 10 was not observed. In an illustrative embodiment, arearrangement has been limited to the use of N-tosylisocyanate, but notothers. (Id.). This was further corroborated by the facile ring-closureof 9 to form 3. All intermediates and final product were fullycharacterized by spectroscopic and analytical data. (See ExampleSection).

The core tricyclic structure of Compound 3 containing 14π electrons isaromatic by the Hückel rule. Nevertheless, with six nitrogen atoms and aconjugated carbonyl group present in the heterocyclic ring system,Compound 3 is considerably electrophilic. In order to explore thisaspect a little further, Compound 3 was reacted with a few carbon andnitrogen nucleophiles. Illustrative examples of the carbon nucleophilesattempted include anisole, N,N-dimethylaniline, and1,2,3-trimethoxybenzene, all of which contain electron-donatingsubstituent(s) on their aromatic rings. Thus, the reaction of a mixtureof 3 (1 mmol), anisole (5 mL), and TFA (10 mL) at 60° C. for 3 h (Scheme3) formed a novel product 11 which was isolated, purified (81%) andcharacterised.

In order to elucidate the pathway of formation of 11 from 3, the latter(1 mmol) was treated with a mixture of TFA (10 mL) and anisole (5 mL) 16at rt for 12 h (Scheme 4), which yielded a mixture of 11 (10%) and 12(60%) which was found to be an adduct of anisole by spectroscopic andanalytical data. Surprisingly, 12 was converted into 11 when heated withTFA at 60° C. for 1 h. This suggests that the reaction proceeds by firstaddition of anisole, followed by selective cleavage of the N-3 PMBgroup. As TFA is often used to remove the PMB group from heterocyclicrings, the observed deprotection of one of the imidazole rings underthese conditions is not totally surprising. (See, e.g., Miki, Y.;Hachiken, H.; Kashima, Y.; Sugimura, W.; Yanase, N. Heterocycles 1998,48, 1).

The generality of the above reaction was studied using two otherelectron-rich carbon nucleophiles, including N,N-dimethylaniline and1,2,3-trimethoxybenzene. Thus, when 3 (1 mmol) was heated separately(Scheme 5) at 60° C. for 6 h with N,N-dimethylaniline (5 mL) or1,2,3-trimethoxy benzene (5 mL) in TFA (10 mL), compound 13 (78%) or 14(80%), was formed, respectively.

In certain illustrative embodiments, the structures of 3 and 11-14 weredetermined by 1D and 2D NMR experiments (some important peaks andcorrelations are presented in FIG. 2 with an example of 12; and Tables1-3), including HMQC, HMBC and DEPT experiments. In the HMBC spectra,H-5 showed the correlations with C-6a and C-11; H-1′ showed correlationwith C-8, C-3′ and C-7′; C-8 showed correlations with H-1′. The additionof anisole/1,2,3-trimethoxybenzene/N,N-dimethylaniline at position 9awas determined by the correlation of H-2″(or/and) and H-6″ with C-9a;two-bond coupling enhancement between C-9a and H-9 (N) and H-9 (N) andC-8.

Expression of DDX3

DDX3 is overexpressed in a number of different types of cancers.However, normal tissue does not typically express DDX3. It has beenshown that cell viability in cell lines is reduced upon treatment withcompounds that downregulate DDX3 or downregulate DDX3 expression.Significantly, as a result of this differential expression, compoundsthat target DDX3, such as those disclosed herein and in particularCompound 3, show lower toxicity to normal cells than typicalchemotherapeutic agents. Specifically, cancer cell lines with highexpression of DDX3 were shown to be responsive to Compound 3. Thus, thepresence of tumor cells that express DDX3 can provide particularlyuseful treatment options, which is indicative for DDX3 to be apredictive biomarker.

The differential expression of DDX3 also provides for a biomarker thatcan have prognostic value with respect to detection of cancerous cellsthat could lead to a disease state. That is, if a tissue sample, forexample a sample from a subject, is found to include cells that expressDDX3, this makes it likely that those cells within the tissue could leadto a disease state even if the disease itself is not yet detected. Itwas also found that the expression of DDX3 is proportional to cancerseverity. More aggressive forms of cancer, for example more aggressiveforms of breast cancer, have been shown by immunohistochemistry and byqRT-PCR to express DDX3 at levels higher than found in less aggressivecell types. Similarly, it has been found that as cancer severityincreases, the expression of DDX3 also increases. This has also beenshown by immunohistochemistry (IHC) and by qRT-PCR. The same resultshave been found in sample tissues. The differential expression of DDX3thus provides for a biomarker that can have prognostic value withrespect to progression of a disease state. Thus, assessment of thedegree of or changes in the degree of expression of DDX3 can be anindicator of cancer severity or progress.

In accordance with the invention, DDX3 expression as measured by, forexample, IHC or qRT-PCR analysis of tissues, can provide both aprognostic and a predictive role in cancer therapy. The observedexpression of DDX3 in various cancers and its utility is described belowand in the non-limiting examples that follow.

Breast Cancer

DDX3 expression was investigated by IHC in breast cancer tissue of 540patients. Clinicopathological characteristics like age, tumor size,lymph node involvement, BRCA1 mutation status, tumor type and grade,mitotic activity index (MAI) and expression of ERα, PR, HER2, Ki-67,cyclin A, cyclin D1, p21, p27, p53, FANCD2 and EGFR were correlated withDDX3 expression and prognostic value was tested.

High cytoplasmic DDX3 expression was observed in 31% of breast cancersamples which was associated with a ductal phenotype (p=0.000), highgrade (p=0.007), HER2 positive (p=0.001) and high MAI (p=0.004). Higherexpression of DDX3 was observed in metastatic disease and theHER2-driven breast cancer subtype. Nuclear expression of DDX3 was shownin 12% of samples and had a negative correlation with intensity ofcytoplasmic DDX3 expression (R=−0.183 p=0.012). The 5-year survival ratewas 87.4% in low cytoplasmic DDX3 expressing tumors compared to 78.9% intumors with high DDX3 expression (p=0.042).

Cytoplasmic overexpression of DDX3 in human breast cancer is associatedwith a more aggressive phenotype, indicating an oncogenic role for DDX3in breast cancer. DDX3 expression could not only be used as a prognosticmarker in breast cancer but could also be an excellent biomarker fortreatment aimed to abrogate DDX3 function.

Lung Cancer

Lung cancer is the most common cancer worldwide, more importantly, onlyabout 30% of non-small-cell lung cancer (NSCLC) patients respond tocurrent combination therapy. DDX3 expression levels of 95 lung cancerpatients was assessed. All lung tumor samples showed elevated levels ofDDX3 but no DDX3 expression was observed in normal lung tissue. Of alllung cancer patients, 53% had high expression of DDX3 and in advancedlung cancer as much as 67% of patients had high expression of DDX3.

Accordingly, high cytoplasmic expression of DDX3 is correlated with anaggressive phenotype of lung cancer. Moreover, all lung tumors hadelevated levels of DDX3. This indicates a predictive role of DDX3expression, assessed by IHC, in the treatment allocation of lung cancerwith DDX3 inhibitors.

Brain Cancers

Several different types of brain cancer have been independently assessedfor DDX3 expression as described below:

Medulloblastoma is the most common malignant brain tumor of childhood.About 24% of tumors have high DDX3 expression based on IHC of 289medulloblastoma tumors, from both pediatric and adult patients.Especially in the WNT sub group of medulloblastoma, either the DDX3Xgene is mutated or DDX3 is overexpressed in about an equal amount ofinstances.

Pontine carcinoma is a rare form of childhood brain cancer. Nonetheless,mortality is a 100% and treatment is warranted. We have assessed 50pontine tumors of which some showed a different expression pattern ofDDX3. Although analysis of this data is still in progress these findingscould lead to specific anti-DDX3 treatment in selected pontine tumorpatients, based on their IHC staining pattern of DDX3.

Glioblastoma is the most common form of adult brain cancer with anabysmal morbidity and mortality and very limited treatment options. Weassessed DDX3 expression In 211 glioblastoma patients, 56% had highexpression of DDX3 indicating that DDX3 expression is a viable biomarkerfor glioblastoma.

Prostate Cancer

Prostate cancer is the most common cancer in man. DDX3 expression wasevaluated in 71 prostate cancer samples. A strong correlation wasobserved between DDX3 and cell proliferation biomarker Ki-67 (R=0.332;p<0.001), which has been shown to be significantly associated withsurvival in prostate cancer (Bettencourt et al., 1996). Also, there is atrend in higher levels of DDX3 in tumors which were Gleason grade 7 orhigher (OR=2.133). This is in line with previous data, which shows asignificant lower survival in breast cancer patients with highexpression of DDX3 (survival rate in low DDX3 expression=87.4% vs. 78.9%in high DDX3 expressing patients; p=0.042). This indicates a potentialrole for DDX3 in the pathogenesis of human prostate cancer. As in breastcancer, DDX3 expression could be a prognostic as well as a predictivebiomarker.

Kidney Cancer

In 140 kidney tumor samples and 140 matched normal kidney samplesassessed for DDX3 expression, there was an increased expression of DDX3by IHC compared to normal tissue. Further analyses ofclinicopathological features are in progress. Based on the increasedexpression of DDX3 in kidney tumors, DDX3 is a predictive marker foranti-DDX3 treatment in kidney cancer.

Sarcoma

A total of 158 sarcoma samples (15 subtypes—chondrosarcoma, malignantfibrous histiocytoma (MFH), clear cell sarcoma, malignant peripheralnerve sheath tumor (MPNST), epithelioid sarcoma, myxoid sarcomas, EwingsSarcoma (ESFT or PNET), Kaposi's sarcoma, leiomyosarcoma, pleomorphicsarcoma, fibrosarcoma, low-grade sarcoma and rhabdomyosarcoma (RMS) wereanalyzed for DDX3 expression by IHC. There was an increased expressionof DDX3 by IHC in about 97% of cancer samples as compared to normaltissue. Only 5 samples (3%) had no DDX3 expression. Eighty-six samples(54%) had moderate to strong expression, and 67 samples (42%) had weakexpression. These data support a role for DDX3 in sarcoma biogenesis andshow that DDX3 expression could be used as a predictive biomarker fortreatment with compounds that downregulate DDX3 expression such asCompound 3. Further analyses of clinicopathological features are inprogress. Based on the increased expression of DDX3 in sarcomas, DDX3 isa predictive marker for anti-DDX3 treatment in sarcomas.

Therapeutic Uses of the Compounds of the Invention

In accordance with the invention, a composition or formulationcomprising a compound that downregulates expression of DDX3 andoptionally a pharmaceutically acceptable vehicle, is administered to asubject, particularly a mammal, for example a human, experiencing one ormore of the following disorders: conditions caused by uncontrolled cellgrowth, hyperproliferation of cells, tumor growth, and cancers. In someembodiments, the cancer is breast cancer; lung cancer; prostate cancer;brain cancer including glioblastoma, medulloblastoma, and pontinetumors; kidney cancer; leukemia; or a sarcoma. Sarcomas includechondrosarcoma, malignant fibrous histiocytoma (MFH), clear cellsarcoma, malignant peripheral nerve sheath tumor (MPNST), epithelioidsarcoma, myxoid sarcomas, Ewings Sarcoma (ESFT or PNET), Kaposi'ssarcoma, leiomyosarcoma, pleomorphic sarcoma, fibrosarcoma, low-gradesarcoma and rhabdomyosarcoma (RMS). In embodiments, the disorder is onewhich has been previously identified as expressing DDX3 and thussusceptible to treatment by down regulation of DDX3. In someembodiments, the cancer is lung cancer. In some embodiments, the canceris breast cancer. In some embodiments, the cancer is prostate cancer. Insome embodiments, the cancer is glioblastoma

In accordance with the invention, a composition or formulationcomprising a fused diimidazodiazepine ring compound of the invention andoptionally a pharmaceutically acceptable vehicle, is administered to amammal, preferably a human, experiencing one or more of the followingdisorders: conditions caused by uncontrolled cell growth,hyperproliferation of cells, tumor growth, and cancers.

The invention also encompasses methods for treating breast cancer; lungcancer; prostate cancer; brain cancer including glioblastoma,medulloblastoma, and pontine tumors; kidney cancer; leukemia; or asarcoma, which comprises administering to a mammal in need of suchtreatment or prevention a therapeutically or prophylactically effectiveamount of a composition comprising a compound that downregulates DDX3,or a pharmaceutically acceptable salt or prodrug thereof, and apharmaceutically acceptable vehicle.

The invention also encompasses methods for treating or preventing breastcancer; lung cancer; prostate cancer; brain cancer includingglioblastoma, medulloblastoma, and pontine tumors; kidney cancer;leukemia; or a sarcoma, which comprises administering to a mammal inneed of such treatment or prevention a therapeutically orprophylactically effective amount of a composition comprising a fuseddiimidazodiazepine ring compound of Formula I-V, or a pharmaceuticallyacceptable salt or prodrug thereof, and a pharmaceutically acceptablevehicle.

In certain embodiments, a composition or formulation comprising acompound of Formula I-V is useful in treating or preventing conditionscaused by uncontrolled cell growth.

In certain embodiments, a composition or formulation comprising acompound of Formula I-V is useful in killing abnormal or cancerous cellswhile simultaneously not affecting healthy or normal cells.

In certain embodiments, a composition or formulation comprising acompound of Formula I-V acts as a cytotoxic agent.

In certain embodiments, a composition or formulation comprising acompound of Formula I-V acts as apoptotic agent.

In one embodiment, “treatment” or “treating” and the like refers to anamelioration of a disease or disorder, or at least one discerniblesymptom thereof. In another embodiment, “treatment” or “treating” refersto an amelioration of at least one measurable physical parameter, notnecessarily discernible by the patient. In yet another embodiment,“treatment” or “treating” refers to inhibiting the progression of adisease or disorder, either physically, e.g., stabilization of adiscernible symptom, physiologically, e.g., stabilization of a physicalparameter, or both. In yet another embodiment, “treatment” or “treating”refers to delaying the onset of a disease or disorder.

In certain embodiments, the compositions of the invention areadministered to a patient, preferably a human, as a preventative measureagainst such diseases. As used herein, “prevention” or “preventing”refers to a reduction of the risk of acquiring a given disease ordisorder. In a preferred mode, the compositions of the present inventionare administered as a preventative measure to a patient.

Therapeutic/Prophylactic Administration of Compositions and Formulations

Due to the activity of the compounds that downregulate DDX3 expression,for example the fused diimidazodiazepine ring compounds of theinvention, such compounds are advantageously useful in veterinary andhuman medicine. As described above, these compounds of the invention areuseful for the treatment or prevention of conditions caused byuncontrolled cell growth, hyperproliferation of cells, tumor growth, andcancers, for example, breast cancer; lung cancer; prostate cancer; braincancer including glioblastoma, medulloblastoma, and pontine tumors;kidney cancer; leukemia; or a sarcoma. Include chondrosarcoma, malignantfibrous histiocytoma (MFH), clear cell sarcoma, malignant peripheralnerve sheath tumor (MPNST), epithelioid sarcoma, myxoid sarcomas, EwingsSarcoma (ESFT or PNET), Kaposi's sarcoma, leiomyosarcoma, pleomorphicsarcoma, fibrosarcoma, low-grade sarcoma and rhabdomyosarcoma (RMS). Inembodiments, compounds that downregulate DDX3 expression are useful forthe treatment or prevention of conditions caused by uncontrolled cellgrowth, hyperproliferation of cells, tumor growth, and cancers, forexample, lung cancer, pancreatic cancer, leukemia, breast cancer, livercancer, kidney cancer, human glioblastoma and prostate cancer.

The invention provides methods of treatment and prophylaxis byadministering to a subject a therapeutically effective amount of acomposition that includes a compound that downregulates DDX3 expression,for example a fused diimidazodiazepine ring compound as disclosedherein. The patient is a mammal, including, but not limited, to ananimal such a cow, horse, sheep, pig, chicken, cat, dog, mouse, rat,rabbit, guinea pig, and is more preferably a human.

The present compositions, which comprise one or more compounds of theinvention, are preferably administered intravenously or orally.

However, suitable dosage ranges of the compounds of the invention aregenerally about 0.0001 milligram to 2000 milligrams of a compound of theinvention per kilogram body weight. In specific preferred embodiments ofthe invention, the oral dose is about 0.001 milligram to about 1500milligrams per kilogram body weight, more preferably about 0.01milligram to about 1000 milligrams per kilogram body weight, morepreferably about 0.1 milligram to about 500 milligrams per kilogram bodyweight, and yet more preferably about 1 milligram to about 100milligrams per kilogram body weight.

The compounds of the invention may also be administered by any otherconvenient route, for example, by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with another biologically active agent. Administration can besystemic or local. Various delivery systems are known, e.g.,encapsulation in liposomes, microparticles, microcapsules, capsules,etc., and can be used to administer a compound of the invention. Incertain embodiments, more than one compound of the invention isadministered to a patient. Methods of administration include but are notlimited to intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, oral, sublingual, intranasal,intracerebral, intravaginal, transdermal, rectally, by inhalation, ortopically, particularly to the ears, nose, eyes, or skin. The preferredmode of administration is left to the discretion of the practitioner,and will depend in-part upon the site of the medical condition. In mostinstances, administration will result in the release of the compounds ofthe invention into the bloodstream.

In specific embodiments, it may be desirable to administer one or morecompounds of the invention locally to the area in need of treatment.This may be achieved, for example, and not by way of limitation, bylocal infusion during surgery, topical application, e.g., in conjunctionwith a wound dressing after surgery, by injection, by means of acatheter, by means of a suppository, or by means of an implant, saidimplant being of a porous, non-porous, or gelatinous material, includingmembranes, such as sialastic membranes, or fibers. In one embodiment,administration can be by direct injection at the site (or former site)of an atherosclerotic plaque tissue.

Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent, or viaperfusion in a fluorocarbon or synthetic pulmonary surfactant. Incertain embodiments, the compounds of the invention can be formulated asa suppository, with traditional binders and vehicles such astriglycerides.

In another embodiment, the compounds of the invention can be deliveredin a vesicle, in particular a liposome (see Langer, 1990, Science249:1527-1533; Treat et al., in Liposomes in the Therapy of InfectiousDisease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York,pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.).

In yet another embodiment, the compounds of the invention can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.14:201; Buchwald et al., 1980, Surgery 88:507 Saudek et al., 1989, N.Engl. J. Med. 321:574). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol.Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989,J. Neurosurg. 71:105). In yet another embodiment, a controlled-releasesystem can be placed in proximity of the target of the compounds of theinvention, e.g., the liver, thus requiring only a fraction of thesystemic dose (see, e.g., Goodson, in Medical Applications of ControlledRelease, supra, vol. 2, pp. 115-138 (1984)). Other controlled-releasesystems discussed in the review by Langer, 1990, Science 249:1527-1533)may be used.

The present compositions will contain a therapeutically effective amountof a compound of the invention, optionally more than one compound of theinvention, preferably in purified form, together with a suitable amountof a pharmaceutically acceptable vehicle so as to provide the form forproper administration to the patient.

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “vehicle” refers to a diluent, adjuvant, excipient, or carrier withwhich a compound of the invention is administered. Such pharmaceuticalvehicles can be liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. The pharmaceuticalvehicles can be saline, gum acacia, gelatin, starch paste, talc,keratin, colloidal silica, urea, and the like. In addition, auxiliary,stabilizing, thickening, lubricating and coloring agents may be used.When administered to a patient, the compounds of the invention andpharmaceutically acceptable vehicles are preferably sterile. Water is apreferred vehicle when the compound of the invention is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid vehicles, particularly forinjectable solutions. Suitable pharmaceutical vehicles also includeexcipients such as starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The present compositions, if desired, canalso contain minor amounts of wetting or emulsifying agents, or pHbuffering agents.

The present compositions can take the form of solutions, suspensions,emulsion, tablets, pills, pellets, capsules, capsules containingliquids, powders, sustained-release formulations, suppositories,emulsions, aerosols, sprays, suspensions, or any other form suitable foruse. In one embodiment, the pharmaceutically acceptable vehicle is acapsule (see e.g., U.S. Pat. No. 5,698,155). Other examples of suitablepharmaceutical vehicles are described in “Remington's PharmaceuticalSciences” by A. R. Gennaro.

In another embodiment, the compounds of the invention are formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compounds of the invention for intravenous administration are solutionsin sterile isotonic aqueous buffer. Where necessary, the compositionsmay also include a solubilizing agent. Compositions for intravenousadministration may optionally include a local anesthetic such aslignocaine to ease pain at the site of the injection. Generally, theingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where the compound ofthe invention is to be administered by infusion, it can be dispensed,for example, with an infusion bottle containing sterile pharmaceuticalgrade water or saline. Where the compound of the invention isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

It is preferred that the compositions of the invention be administeredorally. Formulations for oral delivery may be in the form of tablets,lozenges, aqueous or oily suspensions, granules, powders, emulsions,capsules, syrups, or elixirs, for example. Orally administeredcompositions may contain one or more optionally agents, for example,sweetening agents such as fructose, aspartame or saccharin; flavoringagents such as peppermint, oil of wintergreen, or cherry; coloringagents; and preserving agents, to provide a pharmaceutically palatablepreparation. Moreover, where in tablet or pill form, the compositionsmay be coated to delay disintegration and absorption in thegastrointestinal tract thereby providing a sustained action over anextended period of time. Selectively permeable membranes surrounding anosmotically active driving compound are also suitable for orallyadministered compounds of the invention. In these later platforms, fluidfrom the environment surrounding the capsule is imbibed by the drivingcompound, which swells to displace the agent or agent compositionthrough an aperture. These delivery platforms can provide an essentiallyzero order delivery profile as opposed to the spiked profiles ofimmediate release formulations. A time delay material such as glycerolmonostearate or glycerol stearate may also be used. Oral compositionscan include standard vehicles such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,etc. Such vehicles are preferably of pharmaceutical grade.

The amount of a compound of the invention that will be effective in thetreatment of a particular disorder or condition disclosed herein willdepend on the nature of the disorder or condition, and can be determinedby standard clinical techniques. In addition, in vitro or in vivo assaysmay optionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the compositions will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. However, suitable dosage ranges for oraladministration are generally about 0.001 milligram to about 200milligrams of a compound of the invention per kilogram body weight. Inspecific preferred embodiments of the invention, the oral dose is about0.01 milligram to about 100 milligrams per kilogram body weight, morepreferably about 0.1 milligram to about 50 milligrams per kilogram bodyweight, more preferably about 0.5 milligram to about 20 milligrams perkilogram body weight, and yet more preferably about 1 milligram to about10 milligrams per kilogram body weight. In a most preferred embodiment,the oral dose is about 5 milligrams of a compound of the invention perkilogram body weight. The dosage amounts described herein refer to totalamounts administered; that is, if more than one compound of theinvention is administered, the preferred dosages correspond to the totalamount of the compounds of the invention administered. Oral compositionspreferably contain 10% to 95% active ingredient by weight.

In other embodiments, a composition of the invention for oraladministration includes about 0.001 milligram to about 2000 milligramsof a compound of the invention, more preferably about 0.01 milligram toabout 1000 milligrams of a compound of the invention, more preferablyabout 0.1 milligram to about 500 milligrams of a compound of theinvention, and yet more preferably about 1 milligram to about 200milligrams of a compound of the invention.

Suitable dosage ranges for parenteral, for example, intravenous (i.v.)administration are 0.01 milligram to 100 milligrams per kilogram bodyweight, 0.1 milligram to 35 milligrams per kilogram body weight, and 1milligram to 10 milligrams per kilogram body weight. Suitable dosageranges for intranasal administration are generally about 0.01 pg/kg bodyweight to 1 mg/kg body weight. Suppositories generally contain 0.01milligram to 50 milligrams of a compound of the invention per kilogrambody weight and comprise active ingredient in the range of 0.5% to 10%by weight. Recommended dosages for intradermal, intramuscular,intraperitoneal, subcutaneous, epidural, sublingual, intracerebral,intravaginal, transdermal administration or administration by inhalationare in the range of 0.001 milligram to 200 milligrams per kilogram ofbody weight. Suitable doses of the compounds of the invention fortopical administration are in the range of 0.001 milligram to 1milligram, depending on the area to which the compound is administered.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems. Such animal models andsystems are well known in the art.

In other embodiments, a composition of the invention for parenteral, forexample, intravenous administration includes about 0.001 milligram toabout 2000 milligrams of a compound of the invention, more preferablyabout 0.01 milligram to about 1000 milligrams of a compound of theinvention, more preferably about 0.1 milligram to about 500 milligramsof a compound of the invention, and yet more preferably about 1milligram to about 200 milligrams of a compound of the invention.

The invention also provides pharmaceutical packs or kits comprising oneor more containers filled with one or more compounds of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration. In a certain embodiment, the kit contains more than onecompound of the invention.

The compounds of the invention are preferably assayed in vitro and invivo, for the desired therapeutic or prophylactic activity, prior to usein humans. For example, in vitro assays can be used to determine whetheradministration of a specific compound of the invention or a combinationof compounds of the invention is preferred for treating depression, MS,incontinence, or IBS. The compounds of the invention may also bedemonstrated to be effective and safe using animal model systems.

Other methods will be known to the skilled artisan and are within thescope of the invention.

EXAMPLES

It has been surprisingly found that downregulation of DDX3, asdemonstrated, for example, by compounds described herein, can killcancer cells but not affect normal cells at the concentration of thedrug used. It has also been found that DDX3 expression is increased incertain cancers as disease states that can identify disease states andabnormal cells that can be treated by administering a compound thatdownregulates DDX3 expression.

Immunohistochemistry was performed as follows: Sections of 4 μm werecut, transferred on SuperFrost slides (Menzel&Glaeser, Brunswick,Germany), deparaffinized and rehydrated. Endogenous peroxidase was thenblocked for 15 min with a buffer solution containing 0.3% hydrogenperoxide. Antigens were retrieved by boiling for 20 min in 10 mM citratebuffer (pH 6.0), cooled and washed with PBS. Tissue microarrays (TMAs)were incubated in a humidified chamber for 1 hour with polyclonal rabbitanti-DDX3 antibody diluted 1:1000 in PBS/1% BSA/NaN3. Subsequently,sections were washed in PBS and incubated for 30 min with secondaryantibodies (Brightvision, Immunologic, Duiven, The Netherlands), washedwith PBS and developed with diaminobenzidine. Slides were counterstainedwith hematoxylin, dehydrated and cover-slipped.

The invention is illustrated by the following non-limiting examples.

Example 1

The experiments show activation of DDX3 in lung cell lines (FIG. 2A).Moreover, aggressive lung cancer cell lines such as A549 and H23exhibited robust expression of DDX3 (FIG. 2B). Importantly,down-regulation of DDX3 in A549 abrogated the colony forming abilitiesin soft agar assays (FIG. 3). Taken together these data indicate thatDDX3 is one of the components necessary for lung cancer biogenesis andprovides a novel target for lung cancer treatment.

Example 2

DDX3 expression in lung cancer clinical samples. DEAD box helicases havebeen identified as transcriptional co-activators of both growthpromoting and tumor suppressor genes. Thus, we extended our studies toevaluate the expression profile of DDX3 in lung cancer patient samplesby immunohistochemistry. As shown in the photomicrographs of FIG. 4,lung adenocarcinoma sample as well as small cell carcinoma and squamouscell carcinoma showed robust DDX3 expression as compared to the normallung. Normal lung photomicrographs were taken at 40× magnification andthe lung cancer samples at 20×. Interestingly small cell carcinoma andsquamous cell carcinoma had more nuclear staining than theadenocarcinoma sample. This indicates a role for DDX3 in thepathogenesis of human lung cancer.

Example 3

Generating small molecular inhibitors of DDX3 function. As loss of DDX3function abrogated colony forming abilities along with increasedexpression in lung carcinoma samples, a search was initiated to identifymolecules that could target a functional substrate binding site of DDX3protein. Initial work generated a fused diimidazodiazepine ring molecule(Compound 3) to target RNA helicase. To confirm the functional activityof Compound 3 on DDX3, an in vitro assay was carried out to determinethe effects of Compound 3 on helicase activity. DDX3 protein wassynthesized using the pET system from Novagen. Following DDX3purification, the unwinding assays were performed using duplex oligos,purified DDX3 and Compound 3. Quantification was done by evaluating thepixel density of the native duplex oligos and the unwound oligos. Asshown in FIG. 5, 100 μM of Compound 3 was able to reduce unwinding ofthe duplex oligos by at least 60% over a period of 80 minutes. Thisindicates that Compound 3 has the ability to inhibit the unwindingproperties of DDX3.

Example 4

To study the effects of Compound 3 on cell growth and proliferation,lung cancer cell lines (H23 and A549) were used for the initial studies.As shown in FIG. 6, 8 μM of Compound 3 was able to efficiently ablateboth the cancer cell lines. This is the first data set of evaluating theefficacy of a small molecule inhibitor of DDX3 to kill lung cancercells. (See Also FIG. 7.)

Example 5

Effect of hypoxia on the functional activity of Compound 3 to inducecell death. During solid tumor biogenesis, regions of hypoxia developwithin the tumor due to inadequate and poorly formed vasculature. Theseregions have been shown to be resistant to chemo- and radio-therapy andhave also been closely linked to malignant progression. In addition,some have posited that tumor hypoxia is a major limiting factor of thecurability by SABR at lower radiation dose. Most of the currentprescribed chemotherapeutic agents for cancer treatment exhibitinadequacy to induce cell death under hypoxic conditions. To evaluateefficacy of Compound 3 to induce cell death under hypoxic conditions,cells were incubated with CoCl₂ (hypoxia mimetic compound) for 24 hours,following which Compound 3 was added and incubated for 72 hours. Asshown in FIG. 8, Compound 3 was able to retain its activity both undernormoxic and hypoxic conditions, thus making it an excellent agent forcancer treatment in general.

Example 6

The expression profile of DDX3 in breast cancer patient samples byqRT-PCR was studied in order to determine whether DDX3 is lost orover-expressed relative to tumor grade. FIG. 9A illustrates qRT-PCR ofDDX3 levels in a series of immortalized normal breast cell lines (1-2)and breast cancer cell lines (3-7). The breast cancer cell lines are inthe order of aggressive phenotype. FIG. 9B shows the immunoblot analysisfor DDX3 expression in the same cell lines.

As displayed in FIG. 10A, expression levels of DDX3 increase as tumorsprogress from grade 1 to grade 3. There is 1.9 fold increase in DDX3mRNA levels in grade 2 tumors (14 samples) over grade 1 tumors (12samples, P>0.05) and a 4-fold increase in DDX3 mRNA levels in grade 3(13 samples) over grade 2 tumors (P<0.0001). The DDX3 mRNA levels werenormalized with the basal levels of DDX3 from five normal breast tissuesamples. (FIG. 10B shows immunostaining for DDX3 levels in normal humanbreast sections and in breast carcinoma sample.) This indicates thatgain of DDX3 expression correlates with high-grade breast carcinomasamples and can be potentially used as a prognostic marker for breastcancer progression. Similar results were also obtained byimmunohistochemistry on different grades of breast carcinomas. As shownin FIG. 11, grade III breast tumors have high cytoplasmic staining ascompared to grade I. Also, comparison of 423 breast tumor samplesindicated that high cytoplasmic DDX3 expression is associated with highmitotic index and high grade (P=0.05, data not shown). Both sets of dataindicate a functional role for DDX3 in the pathogenesis of human breastcancer.

Example 7

Tumor growth rate in the mammary fat pad of SCID mice (preclinicalbreast cancer model) using wild type and DDX3 knockdown MDA-MB-231 cellsis illustrated in FIG. 12A. FIG. 12B is photomicrographs of crosssection of lungs of animals injected orthotopically (mammary fat pad)with MDA-MB-231 and MDA-MB-231-shDDX3 cells. Note that the lungs fromMDA-MB-231-shDDX3 injected animals showed no tumor formation as comparedto the wild type cells (black arrows points to lung metastasis).

Example 8

MTS Assays were used to evaluate the effective of compounds thatdownregulate DDX3 on viability in a number of different cell lines. Ingeneral, the CellTiter One Solution Assay (Promega) was used. TheCellTiter One Solution Assay is a colorimetric method for determiningthe number of viable cells in proliferation or cytotoxicity assays. TheOne Solution contains MTS compound and an electron coupling reagent PES.The MTS compound is bioreduced by cells into a colored formazan productthat is soluble in tissue culture medium. The quantity of formazanproduct as measured by the amount of 490 nm absorbance is directlyproportional to the number of living cells in culture.

Procedure:

-   -   1. 25,000 cells were seeded in 24 well plate in duplicate for        each concentration of Compound 3.    -   2. Twenty four hours after seeding, the medium was replaced with        Compound 3 in fresh medium. Cells were incubated for 72 hrs.        After completion of the treatment the media containing Compound        3 was replaced with MTS reagent. Culture medium “no-cell”        control was used as a negative control at the same time.    -   3. Absorbance at 490 nm (450-540 nm) was measured using a plate        reader. The absorbance values were subtracted from “no-cell”        control, which yield the corrected absorbance.

MTS assays of MCF 10A cells (immortalized normal breast cell line)incubated with Compound 3 are illustrated in FIG. 13. X-axis indicatesconcentration of drug used. The cells were incubated for three days(fresh drug was added daily) following which cell viability wasdetermined.

MTS assays of MCF 10A cells (immortalized normal breast cell line)incubated with Compound 3 are illustrated in FIG. 14. X-axis indicatesconcentration of drug used. The cells were incubated for three days(fresh drug was added daily) following which cell viability wasdetermined.

MTS assays of MCF-7 cells (breast cancer cell line) incubated withCompound 3 are illustrated in FIG. 15. X-axis indicates concentration ofdrug used. The cells were incubated for three days (fresh drug was addeddaily) following which cell viability was determined.

MTS assays of MDA-MB-468 cells (breast cancer cell line) incubated withCompound 3 are illustrated in FIG. 16. X-axis indicates concentration ofdrug used. The cells were incubated for three days (fresh drug was addeddaily) following which cell viability was determined.

MTS assays of MDA-MB-231 cells (breast cancer cell line) incubated withCompound 3 are illustrated in FIG. 17. X-axis indicates concentration ofdrug used. The cells were incubated for three days (fresh drug was addeddaily) following which cell viability was determined.

MTS assays of HL60T cells (leukemia cell line) incubated with Compound 3are illustrated in FIG. 18. X-axis indicates concentration of drug used.The cells were incubated for three days (fresh drug was added daily)following which cell viability was determined.

MTS assays of HNT34 cells (leukemia cell line) incubated with Compound 3are illustrated in FIG. 19. X-axis indicates concentration of drug used.The cells were incubated for three days (fresh drug was added daily)following which cell viability was determined.

MTS assays of KG101 cells (leukemia cell line) incubated with Compound 3are illustrated in FIG. 20. X-axis indicates concentration of drug used.The cells were incubated for three days (fresh drug was added daily)following which cell viability was determined.

MTS assays of U87 cells (glioblastoma human cell line) incubated withCompound 3 are illustrated in FIG. 21. X-axis indicates concentration ofdrug used. The cells were incubated for three days (fresh drug was addeddaily) following which cell viability was determined.

MTS assays of PC3 cells (prostate cancer cell line) incubated withCompound 3 are illustrated in FIG. 22. X-axis indicates concentration ofdrug used. The cells were incubated for three days (fresh drug was addeddaily) following which cell viability was determined.

Example 9

Prostate cancer samples stained for DDX3 expression showed robust DDX3expression as compared to the normal prostate. In a patient cohort(n=90), a strong correlation was observed with DDX3 and cellproliferation biomarker Ki-67 (R=0.332; p<0.001), which has been shownto be significantly associated with survival in prostate cancer. Also, atrend of higher levels of DDX3 in 35% of tumors which were Gleason grade7 or higher (OR=2.133) was observed and the staining intensity of DDX3in normal vs. cancer cells (Gleason grade 7 or higher) was significant(p<0.02). This illustrates the role of DDX3 in the pathogenesis of humanprostate cancer. Representative photomicrographs of normal tissuesections and prostate carcinoma tissue sections stained with DDX3antibody are shown in FIG. 23.

Example 10

A total of 158 sarcoma samples (15 subtypes) were analyzed for DDX3expression by IHC. The results were as follows:

Degree of DDX3 expression moderate Sarcoma type (N) none weak or strongAngiosarcoma (9) 0 7 2 Chondrosarcoma (4) 0 2 2 Clear cell sarcoma (4) 01 3 Epithelioid sarc. (8) 0 0 8 ESFT/PNET (25) 3 12 10 Leiomyosarcoma(13) 0 9 4 Low-grade sarcoma (3) 0 2 1 Sarcoma type (N) Liposarcoma (12)0 2 10 MFH (17) 0 6 11 MPNST (4) 0 2 2 Myxoid sarcomas (19) 0 11 8Kaposi's sarcoma (27) 2 8 17 Pleo. sarcoma (5) and fibrosarcoma (1) 0 15 RMS (7) 0 4 3

As shown above, only 5 samples (3%) had no DDX3 expression. Eighty-sixsamples (54%) had moderate to strong expression, and 67 samples (42%)had weak expression. Exemplary photomicrographs (chondrosarcoma andpleomorphic sarcoma tissue sections) stained with DDX3 antibody areillustrated in FIG. 24.

Example 11

DDX3 expression was investigated by IHC in brain tumors. FIG. 25 showsexemplary photomicrographs of normal brain tissue, glioblastoma andmedulloblastoma. As shown in FIG. 25, glioblastoma and medulloblastomaexhibit increased expression of DDX3 as compared to normal brain tissue.

Example 12

In order to determine expression of DDX3 in various cancer cell lines,immunoblot analyses were undertaken and compared to normal cell lines.FIG. 24 illustrates immunoblot analyses for DDX-3 expression in normallung cell line (AEF), lung cancer cell lines (H-23 and A546), prostatecancer cell line (PC-3) and glioblastoma cancer cell line U87).

Example 13

Analysis of the toxicity of Compound 3 was conducted in SCID mice. FIGS.25-29 show tissue samples in control and untreated samples of brain(cerebellum), kidney, Liver, spleen, and blood, respectively. Data shownis that of 500 μM of Compound 3 of the invention that were injectedtwice daily for four weeks. Following that the drug was injected once aweek for three weeks. At the end of the experiment the animals weresacrificed and histopathology performed. The range of drug dose testedwas from 500 to 100 μM. An extensive pathological examination of alltissues was performed following necropsy, which indicated no tissuedamage. Subsequently, we carried out pharmacokinetics in multiple tissuecompartments. For this a LC/MS/MS method was developed for determinationof Compound 3 in mouse plasma (FIG. 30—inset). Sample preparationinvolved acetonitrile precipitation and separation of Compound 3 and theinternal standard, temazepam, was achieved on a Waters Xterra column. Asindicated in FIG. 30, brain exposures are well managed and targettissues are well above effective killing concentrations. The animalswere injected I.P. (single dose of 20 mg/kg with five animals per arm)and the tissue harvested at different time intervals (30 minutes, 60minutes and 24 hours) and analyzed.

The results confirm that the illustrative compounds of the inventionwere successful in killing cancer cell lines while at the same time notaffecting normal cells.

The present invention is not to be limited in scope by the specificembodiments disclosed in the examples which are intended asillustrations of a few aspects of the invention and any embodimentswhich are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art and are intended to fall within the appended claims.

A number of references have been cited, the entire disclosures of whichare incorporated herein by reference.

What is claimed is:
 1. A method of identifying a subject having ahyperproliferative disorder susceptible to treatment by down regulationof DDX3 comprising: a) obtaining a cell or tissue sample from tissue ofthe subject associated with or suspected to be associated with thedisorder; b) optionally isolating cells from the tissue; c) measuringexpression of DDX3 in the tissue or in the cells isolated from thetissue using qRT-PCR and the PCR primers and probes specific for DDX3,or using immunohistochemistry and antibodies specific for DDX3 or anantigen binding fragment thereof; d) providing a referencenon-neoplastic tissue sample; e) comparing the level of expression ofthe DDX3 gene from the tissue sample of the subject, to the level ofexpression of the DDX3 gene of in a reference non-neoplastic tissuesample; and f) identifying the subject as having the hyperproliferativedisorder as susceptible to treatment by down regulation of DDX3 if DDX3is expressed at an increased level in the tissue or cell sample of thesubject when compared to the reference non-neoplastic tissue sample. 2.The method of claim 1, further comprising administering to the subjectan effective amount of a composition or formulation comprising acompound that downregulates DDX3.
 3. The method of claim 2, wherein thecomposition or formulation comprising a compound of Formula (I) thatdownregulates DDX3, wherein the compound of Formula (I) is:

or pharmaceutically acceptable salts and prodrugs thereof, wherein: R,R′, and R″ are each independently a hydrogen, hydroxyl; substituted orunsubstituted: cyclic or acyclic alkyl group, cyclic or acyclic alkenylgroup, cyclic or acyclic alkynyl group, aryl group, alkylaryl group,arylalkyl group, benzyl group, cyclic and acyclic heteroalkyl group,heteroaryl group; —C(O)R³; —C(S)R³; —S(O)R³; —S(O)₂R³; —C(O)NR³R⁴;—C(S)NR³R⁴; C(S)YR³; —C(O)YR³; -β-D-ribosyl; -α-D-ribosyl; -β-L ribosyl;-α-L-ribosyl; 2′-deoxy-β-D-ribosyl; 2′-deoxy-β-L-ribosyl; 2′-deoxy-α-Dribosyl; 2′-deoxy-α-L-ribosyl; or ribose or deoxyribose sugarssubstituted with one or more halogens; R, R′, and R″ can also form aring with one or more C, S, O, N atoms such that, for example, R and R′together include:

R⁷ is a hydrogen; hydroxyl; substituted and unsubstituted: cyclic andacyclic alkyl group, group, alkenyl group, alkynyl group, aryl group,aryloxy group, alkylary group, aryalkyl group, heteroaryl group,heterocycloalkyl group; —C(O)alkyl; —C(O)alkenyl; —C(O)alkynyl;—C(O)aryl; —C(O)benzyl; —C(O)NR³R⁴; —C(S)alkyl; —C(S)alkenyl;—C(S)alkynyl; —C(S)aryl; —C(S)benzyl; —C(S)NR³R⁴; —C(O)NR³R⁴; —C(S)YR³;—C(O)YR³; wherein - - - - - Q is ═O, ═NH, or ═S; Y is O or S; Z is CH,N, P, or C; — is a single bond or double bond; wherein if — is a doublebond, R² or R⁷ is independently O, S, or NH; n is 1, 2, 3, or 4; R³ andR⁴ are independently a hydrogen; hydroxyl; substituted or unsubstituted:cyclic or acyclic alkyl group, cyclic or acyclic alkenyl group, cyclicor acyclic alkynyl group, aryl group, alkylary group, aryalkyl group,heteroaryl group, heterocycloalkyl group; and r, r′, and r″ are eachindependently an integer from 1 to
 3. 4. The method of claim 3, whereinFormula (I) comprises a compound of Formula (IV):

or pharmaceutically acceptable salts and prodrugs thereof, wherein: R³and R⁴ are each independently a hydrogen; hydroxyl; substituted orunsubstituted: cyclic or acyclic alkyl group, cyclic or acyclic alkenylgroup, cyclic or acyclic alkynyl group, aryl group, alkylary group,aryalkyl group, benzyl group, cyclic or acyclic heteroalkyl group,heteroaryl group; —C(O)R⁵; —C(S)R⁵; —S(O)R⁵; —S(O)₂R⁵; —C(O)NR⁵R⁶;—C(S)NR⁵R⁶; —C(O)YR⁵; —C(S)YR⁵; -β-D-ribosyl; -α-D-ribosyl;-β-L-ribosyl; -α-L-ribosyl; 2′-deoxy-β-D-ribosyl; 2′-deoxy-β-L-ribosyl;2′-deoxy-α-D-ribosyl; 2′-deoxy-α-L-ribosyl; or ribose or deoxyribosesugars substituted with one or more halogens; R⁵ and R⁶ areindependently a hydrogen; hydroxyl; substituted or unsubstituted: cyclicor acyclic alkyl group, cyclic or acyclic alkenyl group, cyclic oracyclic alkynyl group, aryl group, alkylary group, aryalkyl group,heteroaryl group, heterocycloalkyl group; and Y is O or S.
 5. The methodof claim 4, wherein the compound is


6. The method of claim 1, wherein the hyperproliferative disorder isselected from the group consisting of group consisting of breast cancer,lung cancer, prostate cancer, glioblastoma, kidney cancer, leukemia,medulloblastoma, pontine tumors, or a sarcoma.
 7. The method of claim 1,wherein the hyperproliferative disorder is selected from the groupconsisting of breast cancer, lung cancer, prostate cancer orglioblastoma.
 8. The method of claim 1, wherein the hyperproliferativedisorder is breast cancer.
 9. The method of claim 1, wherein thehyperproliferative disorder is lung cancer.
 10. The method of claim 4,wherein the sarcoma is selected from the group consisting ofangiosacrcoma, chondrosarcoma, malignant fibrous histiocytoma (MFH),clear cell sarcoma, malignant peripheral nerve sheath tumor (MPNST),epithelioid sarcoma, myxoid sarcomas, Ewings Sarcoma (ESFT or PNET),Kaposi's sarcoma, leiomyosarcoma, pleomorphic sarcoma, fibrosarcoma,low-grade sarcoma, liposarcoma, and rhabdomyosarcoma (RMS).
 11. Themethod of claim 1, wherein measuring comprises immunohistochemistryanalysis of the tissue sample.
 12. The method of claim 1, furthercomprising isolating cells from the tissue sample and measuringexpression of DDX3 in the tissue comprises measuring expression of DDX3in the cells.
 13. The method of claim 12, further comprising performingan assay by exposing the cells to a compound that downregulates DDX3,measuring cell viability as a function of concentration and identifyingthe hyperproliferative disorder as susceptible to treatment by downregulation of DDX3 if cell viability decreases as a function ofconcentration.
 14. The method of claim 13, wherein the assay comprises aMTS assay.